Activation of central amygdala dynorphin neurons alleviates the affective component of chronic pain

Activation of central amygdala dynorphin neurons alleviates the affective component of chronic pain | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 6 February 2026 V1 Latest version Share on Activation of central amygdala dynorphin neurons alleviates the affective component of chronic pain Authors : Merel Dagher , Flora D’Oliveira da Silva , Ted B. Usdin , Jamie E. Mondello , Gabriella H. Sigal , nicolas massaly , and Catherine Cahill [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.177037587.72835411/v1 267 views 111 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Background and purpose: The neurobiological circuits underlying chronic pain states are not fully elucidated, limiting available treatment options. While the central amygdala (CeA) is a key hub for integrating pain and stress signals, the role of dynorphin-expressing neurons within the CeA remains poorly understood. Experimental approach: We investigated the role of the CeA dynorphin-expressing (CeA dyn ) neurons in the affective and sensory components of chronic neuropathic pain induced by chronic constriction injury. Using dynorphin-Cre mice, we selectively manipulated CeA dyn neuronal activity through chemogenetic, optogenetic and pharmacological approaches. Behavior was assessed using conditioned place preference, real-time place preference paradigms, and measuring mechanical withdrawal thresholds. Finally, whole-brain clearing and anatomical tracing were used to identify projection targets of CeA dyn neurons. Key results: Chemogenetic inhibition of CeA dyn neurons produced a conditioned place aversion in mice with chronic neuropathic pain but had no effect in sham animals. Conversely, optogenetic activation of CeA dyn neurons induced place preference in neuropathic pain mice, with no effect in sham controls. Importantly, neither manipulation altered mechanical withdrawal thresholds. Pharmacological blockade of KOR signaling abolished the place preference associated with optogenetic activation of CeA dyn neurons in neuropathic pain mice. Notably, KOR antagonism revealed a place preference in sham animals, suggesting that dynorphin signaling from CeA dyn neurons is aversive in the absence of chronic pain. In contrast, inhibition of the entire CeA reduced mechanical withdrawal thresholds in neuropathic pain without producing place preference. Anatomical tracing revealed that CeA dyn neurons project to multiple brain regions implicated in affective and pain-related behaviors. Conclusions and Implications: These findings indicate that CeA dyn neurons selectively regulate the affective, but not sensory, component of neuropathic pain. In this context, dynorphin release from CeA dyn neurons appears to be beneficial by alleviating the affective dimension of the pain experience. Activation of central amygdala dynorphin neurons alleviates the affective component of chronic pain Merel Dagher 1* , Flora D’Oliveira da Silva 1* , Ted B. Usdin 2 , Jamie E. Mondello 1 , Gabriella H. Sigal 3 , Nicolas Massaly 4,5 , Catherine M. Cahill 1,4 1 Department of Psychiatry & Biobehavioral Sciences, Hatos Center for Neuropharmacology, Semel Institute for Neuroscience and Human Behavior, University of California Los Angeles, Los Angeles, California, USA, 90095 2 NIMH Intramural Research Program, NIH Main Campus, Bethesda, Maryland 3 Department of Integrative Biology and Physiology, University of California, Los Angeles, Los Angeles, California, USA, 90095 4 Neuroscience Interdepartmental Program, Brain Research Institute, University of California Los Angeles, Los Angeles, California, USA, 90095 5 Department of Anesthesiology and Perioperative Medicine, University of California, Los Angeles, Los Angeles, California, USA, 90095 * Denotes equal author contribution Correspondence to: Merel Dagher at [email protected] Flora D’Oliveira Da Silva at [email protected] Catherine M Cahill at [email protected] ORCID ID: Merel Dagher: 0000-0003-0325-4543 Flora D’Oliveira Da Silva: 0009-0008-3708-4988 Ted Usdin: 0000-0003-3813-0049 Jamie E. Mondello: 0000-0003-0103-4897 Gabriella H. Sigal: 0009-0009-7215-9364 Nicolas Massaly: 0000-0003-3440-5957 Catherine M. Cahill: 0000-0001-6936-5524 Abstract Background and purpose: The neurobiological circuits underlying chronic pain states are not fully elucidated, limiting available treatment options. While the central amygdala (CeA) is a key hub for integrating pain and stress signals, the role of dynorphin-expressing neurons within the CeA remains poorly understood. Experimental approach: We investigated the role of the CeA dynorphin-expressing (CeA dyn ) neurons in the affective and sensory components of chronic neuropathic pain induced by chronic constriction injury. Using dynorphin-Cre mice, we selectively manipulated CeA dyn neuronal activity through chemogenetic, optogenetic and pharmacological approaches. Behavior was assessed using conditioned place preference, real-time place preference paradigms, and measuring mechanical withdrawal thresholds. Finally, whole-brain clearing and anatomical tracing were used to identify projection targets of CeA dyn neurons. Key results: Chemogenetic inhibition of CeA dyn neurons produced a conditioned place aversion in mice with chronic neuropathic pain but had no effect in sham animals. Conversely, optogenetic activation of CeA dyn neurons induced place preference in neuropathic pain mice, with no effect in sham controls. Importantly, neither manipulation altered mechanical withdrawal thresholds. Pharmacological blockade of KOR signaling abolished the place preference associated with optogenetic activation of CeA dyn neurons in neuropathic pain mice. Notably, KOR antagonism revealed a place preference in sham animals, suggesting that dynorphin signaling from CeA dyn neurons is aversive in the absence of chronic pain. In contrast, inhibition of the entire CeA reduced mechanical withdrawal thresholds in neuropathic pain without producing place preference. Anatomical tracing revealed that CeA dyn neurons project to multiple brain regions implicated in affective and pain-related behaviors. Conclusions and Implications: These findings indicate that CeA dyn neurons selectively regulate the affective, but not sensory, component of neuropathic pain. In this context, dynorphin release from CeA dyn neurons appears to be beneficial by alleviating the affective dimension of the pain experience. Introduction Chronic pain affects approximately 20% of adults worldwide 1 and is strongly associated with psychiatric comorbidities. According to the American Psychological Association, over half of adults suffering from chronic pain report persistent symptoms of anxiety and depression 2 . A recent meta-analysis including patients from 50 countries reported pooled prevalence rates of 39.3% for depression and 40.2% for anxiety among individuals with chronic pain 3 . These observations underscore the tight link between chronic pain and affective disturbances, highlighting the need to understand the neurobiology of the emotional component of chronic pain. The amygdala is a key limbic structure integrating emotional and sensory information through extensive connections with cortical, subcortical, and brainstem regions. 4 Beyond its well-established role in fear, anxiety and affective processing, the amygdala is increasingly recognized as a critical hub for the emotional–affective aspects of pain 5 . Within the amygdala, the central nucleus (CeA) — often referred to as the nociceptive amygdala — receives direct nociceptive input via the spino–parabrachio–amygdaloid pathway 6 . The CeA neurons are activated by noxious stimulation 7 , and this activation is enhanced in neuropathic pain via synaptic plasticity 8 . Moreover, the CeA is primarily comprised of inhibitory GABA-releasing neurons, however has heterogeneous cell subtypes, including those expressing protein kinase C-δ (PKCδ), enkephalin, somatostatin (SST), dynorphin (Dyn), and corticotropin-releasing hormone (CRH). Despite CeA involvement in mediating chronic pain states, the role of CeA neuronal subtypes remains elusive. The dynorphin/kappa opioid receptor (KOR) system is abundant in the CeA and is well known for its role in stress and anxiety-related behaviors 9 10 . While KOR activation produces dysphoric and aversive effects 11 , including those associated with chronic pain 12–14 , the contribution of CeA dynorphin (CeA dyn ) neurons to the affective component of chronic pain remains unclear. The present study aimed to determine the role of CeA dyn neurons in modulating the emotional and sensory dimensions of chronic pain by assessing the behavioral outcomes of their selective activation or inhibition. We also evaluated mechanical sensitivity thresholds during CeA dyn activation and inhibition and used the selective KOR antagonist aticrapant to assess whether the observed behavioral effects were mediated by dynorphin/KOR signaling or GABA release. Finally, we examined the projection targets of CeA dyn neurons, as many of their efferent pathways remain incompletely characterized. While single-cell RNA sequencing studies have revealed cellular heterogeneity within the CeA, and a small number of studies have investigated CeA dyn -associated circuits in the context of itch and pain, a systematic characterization of CeA dyn output projections is still limited. The projection patterns identified here extend existing knowledge and will require further investigation to determine their functional relevance to behavioral phenotypes. Methods Animals Mice were used because they enable genetic and circuit-specific manipulations required to study neural mechanisms of chronic pain. The chronic constriction injury model reproduces key features of human neuropathic pain 15 , and dynorphin-Cre mice allow selective targeting of dynorphin-expressing neurons within the central amygdala. All experimental procedures were approved by the University of California, Los Angeles Chancellor’s Animal Research Council and were conducted in accordance with institutional and national guidelines for the care and use of laboratory animals. Male and female Pdyn-IRES-Cre breeders were obtained from The Jackson Laboratory at eight weeks of age and bred in-house to generate dynorphin Cre⁺ and dynorphin Cre⁻ littermates. Mice were at least seven weeks of age at the time of experimentation. Male and female mice were included in all experiments to account for sex as a biological variable. Sex effects were evaluated for the primary behavioral outcomes, and because no sex differences were observed, data were pooled across sex for all analyses. Animals were housed in groups of two or three per cage on a 12 h reverse light/dark cycle with food and water available ad libitum . Mice were allowed to habituate to the housing environment for one week prior to handling. Animals were randomly assigned to experimental groups and treatment conditions. Handling was performed in the vivarium for one week prior to all surgeries and behavioral testing. All experiments were conducted during the dark phase between 09:00 and 16:00 h. Behavioral testing and data collection were performed by an experimenter blinded to genotype, surgical condition, and treatment group to minimize bias. Drugs and viruses Clozapine N-oxide (CNO; Sigma-Aldrich, St. Louis, MO, USA; catalog no. C0832) was dissolved in 0.9% sterile saline and used for chemogenetic inhibition of dynorphin neurons at a dose of 1 mg kg -1 to minimize anxiolytic 16 effects previously reported at higher doses. The kappa opioid receptor antagonist (S) -aticaprant (also known as LY2456302; Eli Lilly and Company, Indianapolis, IN, USA; reference no. 033589), a highly potent and selective short-acting kappa opioid receptor antagonist, was dissolved in corn oil at a 10 mg kg -1 dose. The following viruses were used for select experiments: Conditioned place preference with dyn-CeA neurons AAV2-hSyn-DIO-hM4D(Gi)-mCherry AAV2-hSyn-DIO-mcherry Addgene 44362 Addgene 50465 Real time place preference with dyn-CeA neurons AAV5-EF1a-dFloxed-hChR2-EYFP Addgene 20298 Conditioned place preference with whole CeA AAV2-hSyn-hM4D(Gi)-mCherry AAV2-hSyn-mCherry Addgene 44360 Addgene114472 Stereotaxic surgeries Mice were anesthetized with isoflurane (IsoFlo®, Zoetis, Parsippany, NJ, USA) and mounted on a Kopf stereotaxic alignment system. Animals received carprofen (Rimadyl®, Zoetis, Parsippany, NJ, USA) at a dose of 5 mg kg -1 prior to the start of the surgery and for two consecutive days post-operatively for analgesia. A small incision was made to expose the skull surface such that bregma and lambda could be visualized. A hole was drilled into the skull overlaying the central amygdala (CeA). Using a pulled glass capillary and a nano-injector, viruses were injected into the CeA (from bregma: AP -1.1, ML 2.8, DV – 4.6) at volumes between 75-150 nL. The capillary was gradually lowered to 0.05 µm below the target DV and left in place for 5 minutes to create a small canal before it was raised to the target DV for virus injection. It was left in place for an additional 5 minutes following the injection and gradually raised to prevent backflow. The incision was closed with 5-0 Vicryl sutures (Ethicon, Somerville, NJ, USA). For optical fiber implantations, the optical fiber (Neurophotometrics, San Diego, CA, USA; 1.25mm ferrule size, 200µm core diameter) was lowered to 0.05 µm above the target DV and solidified in place with dental cement (C&B Metabond, Parkell, Edgewood, NY, USA). The cement was left to dry for 10 minutes before the animal was removed from anesthesia. Animals received post-operative carprofen (5 mg kg⁻¹) and were monitored for three days to ensure no signs of distress or pain. Animals were allowed to recover for at least two weeks before any subsequent surgery. All experiments were performed 3–6 weeks after viral injection to allow for adequate expression. Chronic constriction injury Mice were randomly assigned to either CCI (pain) or sham (no pain) surgery groups; cage mates always received the same surgical condition. Both CCI and sham mice were anesthetized with 2.5% isoflurane (IsoFlo®, Zoetis, Parsippany, NJ, USA) in oxygen. Surgeries lasted no more than 10 minutes. For the CCI group, a 1 cm incision was made in the upper left hind leg and the sciatic nerve was constricted with polyethylene tubing (PE20) cut to 2 mm in length 17 . For the sham group, a 1 cm incision was made in the upper left hind leg, but the sciatic nerve was not exposed nor cuffed. For post-operative analgesia, mice received liquid acetaminophen (acetaminophen oral solution; Tylenol, Johnson & Johnson Consumer Inc., Skillman, NJ, USA; 32 mg ml⁻¹) administered in their food and were monitored for two consecutive days to ensure proper wound healing. Histology Brains were collected one day after the experiments ended. Mice were overdosed with 100mg kg -1 pentobarbital and brains were extracted and stored in -80 degree C until ready to be sectioned. Brains were coronal-sectioned at 30 μm via a cryostat and mounted on poly-L-lysine coated slides. At least 18 sections were collected to span the anterior and posterior CeA. A DAPI stain was then applied to improve histology and brain visualization. The mCherry and GFP fluorophores were visualized via a Leica DM5500 epifluorescent microscope. Mechanical nociceptive thresholds The Ugo Basile electronic von Frey (eVF) was used to assess mechanical withdrawal thresholds. The handheld unbending filament attaches to a detector that records the grams of force when applied to the plantar surface of the mouse ipsilateral hind paw. Less sensitivity to the filament results in higher grams of force and increased sensitivity to the filament results in less grams of force recorded. Each mouse was allowed to habituate to the von Frey chambers for 15 mins at least one day prior to testing. Mice were tested before and after pain and viral surgeries, as well as with or without dynorphin neuron manipulation. Each mouse was tested three times, and the averaged value was used to generate a single data point. All mice were tested before subsequent testing rounds, as opposed to each mouse getting three consecutive tests to avoid sensitization/desensitization to the filament. Conditioned place preference The conditioned place preference (CPP) test was conducted using an unbiased, counterbalanced, three-chamber apparatus. Each box (28 x 28 x 19 cm) was divided into two equal-sized conditioning chambers and a connecting neutral compartment. The two chambers were distinguished with visual (stripes vs. circles on the walls) and tactile (hard wire vs. mesh flooring) cues. Prior to testing, mice were habituated to the chambers for 15 mins and allowed free access to all three compartments. One day following habituation, mice were once again allowed free access to all compartments, this time for 30 mins, and their behavior was recorded using Anymaze. We refer to this as their pre-conditioning day. One to two weeks after pre-conditioning, mice were conditioned once a day to CNO or vehicle for six days, then tested on day seven for the post-conditioning test. During the post-conditioning test, CPP was performed without drug treatment, in which mice were allowed to explore all three compartments for 30 mins. Time spent and distance traveled in each compartment were recorded. Preference scores were calculated using the following formula: [Time in drug-paired chamber – Time in vehicle-paired chamber] – [Time in drug-assigned chamber at preconditioning – time in vehicle-assigned chamber at preconditioning]. Real-time place preference Mice were placed in a custom-made black plexiglass chamber (50.8×30.7×23.9 cm) that had no visual or tactile cues and allowed to explore freely for 20 min. The arena was virtually divided into two equal zones using ANY-maze software, designated as ‘stimulation’ and ‘no-stimulation’. Zone assignment within the room was counterbalanced across animals to avoid spatial bias. Optogenetic stimulation (450–465 nm, 20 Hz, 10 ms pulse width) was delivered through fiber optic implants when animals entered the ‘stimulation’ zone, via an ANY-maze hardware controller interfaced with a Stoelting Ami-2 and a Prizmatix Optogenetics-LED-Dual light source. No stimulation was delivered in the ‘no-stimulation’ zone. Preference and aversion were assessed by comparing time spent in each zone, and locomotor measures (distance traveled, velocity, entries) were extracted using ANY-maze. CeA dyn projections and brain clearing Eight dyn-cre+ mice (four males, four females) were transfected with 73.6 nL of AAV-hSyn-FLEX-mGFP-2A-synaptophysin-mRuby unilaterally in the right CeA. The injection volume was selected after pilot experiments confirmed larger amounts of the virus labeled cells outside of the CeA. The mice were left to recover in their home cages. Two weeks post-surgery, mice were perfused using 4% PFA in 1x PBS and were post-fixed overnight. Fixed brains were cleared using the CUBIC procedure as previously described 18 . Following two weeks of incubation in CUBIC-L with changes every 1-2 days, the brains were rinsed with several changes of PBS, equilibrated in CUBIC-R (adjusted to RI 1.51), and were embedded in 2% agarose and equilibrated in CUBIC-R. Image acquisition was performed in silicone oil (PM125; Clearco) on an Intelligent Imaging Innovations (3i) Axl instrument. A LaVision 0.35 NA immersion objective (nominally 4X; Miltenyi Biotec) was used at an optical zoom that produced images with 2 pixel/micron lateral resolution. Tiled Z-stacks were collected 4-micron intervals in a single channel using excitation with a 561nm laser, a 595/50 emission filter and 500 msec accumulation time per field on a Hamamatsu Fusion BT camera. Following subtraction of camera background from each image, produced by averaging 50 fields collected with no illumination, stitching was performed using Terastitcher 19 . A UNET-based 20 denoising model trained using 50 averaged fields from 4 regions spanning the range of signal intensities and densities was applied to the stitched image. Stitching and denoising were performed using custom scripts implemented on the NIH High Performance Cluster (https://github.com/SNIR-NIMH), available on request. This work utilized the computational resources of the NIH HPC Biowulf cluster (https://hpc.nih.gov). Visualization and rendering of projections were performed in Zeiss Arivis Pro. Statistical analyses Data are presented as mean values with individual data points and SEM. Statistical analyses were performed using GraphPad Prism (version 10). Prior to inferential analyses, data were assessed for normality and homogeneity of variance, and statistical tests were selected accordingly based on the experimental design and comparison of interest. Data were analyzed using two-way ANOVA, paired or unpaired Student’s t tests, or one-sample t tests, as appropriate. When significant main effects or interactions were detected in ANOVA analyses, post hoc multiple-comparison tests were performed using Sidak’s, Tukey’s, or Fisher’s least significant difference tests, as indicated. The data and statistical analyses comply with the recommendations on experimental design and analysis in pharmacology. Animals were excluded from behavioral analyses if histological verification revealed off-target viral expression and/or incorrect optical fiber placement; reported sample sizes reflect only animals with confirmed targeting. Statistical significance was defined as follows: nonsignificant (ns), P > 0.05; * P ≤ 0.05; ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001. Inhibiting the CeA dyn neurons produces aversion without modulating pain sensitivity in CCI mice. To investigate the specific role of CeA dyn neurons in affective and sensory components of pain, we conducted a series of experiments using dynorphin-cre (Dyn-cre) mice. Mice received unilateral intracranial injections of either a Cre-dependent Gi-DREADD or an mCherry-expressing control viral vector, after which they underwent either CCI or sham surgery one week later (Fig. 1A-B). Two weeks after induction of chronic pain, mice underwent a conditioned place preference paradigm. Mice were conditioned with CNO or vehicle once a day for six days and the place preference test was conducted the following day. In the sham mice, inhibition of CeA dyn neurons did not produce a significant preference or aversion, as indicated by no difference in time spent in the CNO- versus vehicle-paired chambers (Fig. 1C) and by preference scores that were comparable to mCherry controls (Fig. 1D). In contrast, in CCI mice, inhibition of CeA dyn neurons induced a conditioned place aversion, where the Gi-DREADD–expressing mice spent more time in the vehicle-paired chamber compared to mCherry control mice (Fig. 1C) and show postconditioning scores significantly lower than zero indicating an aversion for the CNO-paired chamber (Fig 1D). Furthermore, the CPP scores of CCI Gi-DREADD mice were significantly lower than those of sham Gi-DREADD and CCI-mCherry groups (Fig. 1D), as represented by the heat maps (Fig. 1E). We concurrently examined the effect of CeA dyn neuron inhibition on mechanical sensitivity using von Frey (VF) filaments. Administration of CNO did not alter mechanical withdrawal thresholds in mCherry control animals (Fig. 1F). Inhibition of CeA dyn neurons in Gi-DREADD–expressing mice also failed to modify mechanical thresholds in either sham or CCI groups, indicating that the observed place aversion to CeA dyn inhibition cannot be explained by changes in pain hypersensitivity or allodynia. Statistical values are located in Table 1. Activation of CeA dyn neurons produces a preference without modulating pain sensitivity in CCI mice. The aversive nature of CeA dyn neuronal inhibition in chronic pain states suggests that activation of these neurons might be reinforcing in pain states. To test this hypothesis, we injected a Cre-dependent channelrhodopsin (ChR) or mCherry viral vector into the CeA of dyn-cre + mice. Animals then underwent either CCI or sham surgery, prior to undergoing a real-time place preference (RTPP) test. During the test, mice were allowed to move freely within a chamber in which one half was paired with optogenetic activation of CeA dyn neurons, while the other half was not (Fig. 2A). The time spent on each side of the chamber was used to assess preference for the stimulation versus non-stimulation zone. Because CeA dyn neurons co-release GABA along with dynorphin, the rewarding effect of their activation in CCI mice could arise from the release of either GABA or dynorphin. To determine the extent that optogenetic activation of these CeA dyn produced reinforcing effects, we pharmacologically blocked the kappa opioid receptor (KOR) with the short-acting antagonist aticaprant (10 mg kg -1 ) during RTPP (Fig. 2A-B). Each mouse underwent two RTPP sessions — one following vehicle injection and one following aticaprant injection — with the order counterbalanced across subjects to minimize potential order effects. Vehicle-treated CCI mice showed a significant preference for the stimulation-paired side (Fig. 2C-E), as demonstrated by our pilot experiment (Suppl Fig. 1). However, this preference was prevented when KOR signaling was blocked with aticaprant (Fig. 2C-E), indicating that the rewarding effect of CeA dyn neuronal activation in CCI mice is dependent on release of the neuropeptide dynorphin rather than just GABA transmission. Interestingly, KOR blockade not only eliminated the preference in CCI mice but induced a RTPP in sham animals (Fig. 2C-E). As before, mechanical withdrawal thresholds were determined to assess whether the rewarding effect of CeA dyn neuronal activation may attributed to changes in pain hypersensitivity. Mechanical withdrawal thresholds measured at baseline and during optogenetic stimulation revealed no significant modulation of mechanical sensitivity between vehicle and aticaprant treatments, indicating that CeA dyn neuronal activation influences the affective rather than the sensory component of pain (Fig. 2F). Inhibiting the central amygdala is not reinforcing but reduces mechanical sensitivity in chronic pain. Given the lack of effect of manipulating CeA dyn neuronal activity on sensory thresholds in mice with or without chronic pain, we investigated the role of whole CeA inhibition in conditioned place preference (CPP) and mechanical sensitivity. Dynorphin cre- (WT littermates) mice were transfected with a Gi DREADD or mCherry-expressing control viral vector. Following two weeks, animals underwent sham or chronic nerve constriction surgeries. One week later, mice underwent conditioning to CNO (1 mg kg -1 ) and saline, and CPP to CNO was determined after six days of conditioning (Fig. 3A). Viral placement was validated post experiment completion (Fig. 3B) and only animals with precise placement were included in the final behavioral analysis. No differences were observed between time spent in vehicle or CNO chambers for sham and CCI mice (Fig. 3C). No differences in CPP scores were evident between groups, however CNO produced a CPP in the CCI Gi DREADD mice (Fig. 3D). Administration of CNO had opposing effects on mechanical pain hypersensitivity (Fig 3E), where CNO increased mechanical withdrawal thresholds in sham mice but attenuated them in CCI mice. Projections of CeA dyn neurons To investigate possible circuits involved in the observed behavior, we traced CeA dyn projections using a unilaterally injected cre-dependent virus. Using whole brain clearing, we reconstructed a sagittal view of areas that received inputs from the CeA dyn projections (Fig. 4A). Our data suggests that projections from the CeA dyn go to the striatum and lateral hypothalamus (Fig. 4B), CA3 stratum oriens of the hippocampus (Fig. 4B, C), thalamus and entorhinal cortex (Fig. 4D), and locus coeruleus and parabrachial nucleus (Fig. 4E). These identifications were based on closely examining reconstructed coronal section anatomy and aligning them with brain atlas plates, as well as assessing existing literature of projections already identified. The plates used from the Allen Brain Atlas are as follows: Plate 74, 78, 89, and 109. While some of these projections have been identified, some are novel and require further analysis to be confirmed. Supplemental videos of the whole brain projections have been uploaded (Suppl. Fig. 2). Discussion/Conclusion Our study identified that CeA dyn neurons modulate the affective, but not the sensory, component of chronic pain. Given that CeA dyn neurons are GABAergic projection neurons that also express the endogenous opioid peptide dynorphin, it was important to determine whether the observed behavioral effects were mediated by GABAergic transmission or by dynorphin signaling. To address this, we showed that systemic inhibition of KORs during CeA dyn activation completely abolished the real time place preference observed in mice with chronic pain. These findings demonstrate that dynorphin signaling through KORs is necessary for the induction of this affective pain–related behavioral outcome. While activation of CeA dyn neurons may also engage GABAergic mechanisms, our data specifically identify dynorphin–KOR signaling as a necessary component underlying the affective modulation of chronic pain. Given that manipulation of the CeA dyn neurons did not modify sensory thresholds in either sham or neuropathic pain animals, we conducted experiments with whole CeA inhibition via chemogenetic studies. This inhibition confirmed previous studies that the CeA can modulate the sensory component of chronic pain 21 22 23 . This is potentially mediated by the CeA-PKCδ neurons, as these neurons are pronociceptive 24 , whereas the CeA-somatostatin cells are antinociceptive 25 . Interestingly, inhibiting the CeA did not produce a conditioned placed preference or aversion either in sham or neuropathic pain animals. The CeA is a highly heterogeneous structure with different cell subtypes 26 , where a recent single cell RNA-sequencing study combined with integrated retrograde axonal training with EASI-FISH identified CeA output targets 27 . Pdyn and sst transcripts are highly colocalized in the CeA 28 , as are the pdyn and crf neurons. While the CeA-somatostatin neurons produce the antinociceptive phenotype observed following nerve injury and have been shown to modulate stress-induced insomnia 29 and defensive behaviors 30 , they have not been investigated in the context of affective components of chronic pain. We therefore posit that the dynorphin release from the pdyn/sst subpopulation is necessary for attenuating the aversive pain experience, though may not be sufficient. Importantly, the present study did not directly assess anxiety- or depressive-like behaviors, and thus it remains unclear whether CeA dyn neurons are involved in regulating negative affect per se, as opposed to specifically modulating pain-related aversion. Regarding the pdyn/crf subpopulation, Neugebauer et al. 31 showed that activation of CeA-CRF neurons induced increased vocalizations and anxiety-like behavior, but did not have an effect on mechanical sensitivity in a neuropathic pain model. Moreover, silencing the CeA-CRF neurons decreased vocalization and reduced anxiety-like behavior. The same group showed that intra-CeA KOR 32 activation by U-69,593 produces anxiety-like behavior, increases vocalizations to a noxious stimulus, and increases activity of CeA-CRF neurons through synaptic disinhibition. In contrast, our findings show that activation of CeA dyn neurons reduces pain-associated aversion in neuropathic mice, an effect that is directionally opposite to that reported following activation of CeA-CRF neurons, though the behavioral outcomes are different. Systemic antagonism of KORs attenuates the positive valence associated with CeA dyn activation in pain mice, while producing a significant real-time preference in sham mice. It remains unclear why the antagonist is producing different effects in the sham vs chronic pain state, but it is possible that there is a shift in the dominance of CRF versus dynorphin driving affective states, and this altered ratio engages KORs in the chronic pain state. Alternatively, local CeA KOR is not activated by release of dynorphin from CeA dyn neurons, which could explain the discrepancy in findings. Indeed, there are many dynorphin projection neurons that are a source of peptide to activate CeA KOR including the insula cortex 33 , lateral hypothalamus 34 and substantia nigra 35 . The insula-amygdala circuit was identified to be important in behavioral responses during foraging when facing different choices 36 . It was also suggested to contribute to mood and substance use disorders, where dysregulation of this system may contribute to negative affective states 36 . Dynorphin neurons from each of these brain regions that project to the central amygdala may modulate threat discrimination and can help to dampen anxiety-like behaviors 37 . We also visualized the CeA dyn projections to inform future studies of circuits that may be mediating the aversive component of pain. Using a whole brain imaging, we identified that most if not all CeA dyn neurons project to brain regions outside the CeA. Some of the most intense innervation was found in the parabrachial nucleus, which is not surprising considering the previous extensive and critical reciprocal projections between these brain regions that are involved in aversive information and pain 38–42 . Further, previous studies reported that CeA dyn neurons project to the parabrachial nucleus and optogenetic activation of this specific projection suppressed itch responses 43 . Moreover, the CeA dyn project to the locus coeruleus (LC) and 30% of LC norepinephrine-expressing neurons target the CeA dyn/crf neurons 44 . Another prominent projection was to the caudate nucleus. Projections from the CeA to the striatum were previously shown to be involved in associative learning 45–47 and thus may be an important source of dynorphin to activate KORs located in medium spiny neurons in the direct pathway known to be involved in habit and reward. While KORs in the CeA were previously reported to contribute to the aversive component of the pain experience, it is unlikely that we are specifically engaging these circuits given that the CeA dyn neurons appear to be primarily projecting outside the CeA. Nevertheless, we cannot discount this possibility where Navratilova et al. 48,49 , reported that the CeA-KOR signaling is necessary for pain-induced negative affect. The researchers found that gabapentin place preference is blocked in a SNI-pain model after right-CeA NorBNI, a long-lasting KOR antagonist. Additionally, inactivation of oprk1 in the CeA increases anxiety-like behavior and impairs conditioned threat discrimination 50 . However, reduction of CeA pdyn had no effect on anxiety or conditioned threat discrimination suggesting that these neurons are likely not responsible for the CeA KOR mediated effects. Although, dynorphin inputs to the CeA likely promote threat discrimination and dampen anxiety that could contribute to pain aversion and negative affect associated with pain. From a clinical perspective, these findings suggest that selectively engaging dynorphin-dependent circuits within the central amygdala may represent a strategy to alleviate the affective burden of chronic pain without directly altering sensory pain thresholds. Such circuit-specific approaches could complement existing analgesic treatments by targeting the emotional distress associated with chronic pain, which is often resistant to conventional therapies. Importantly, our results highlight the necessity of precise, cell-type–specific modulation of the dynorphin/KOR system, as global manipulation of this pathway is well known to produce aversive effects. In conclusion, our data supports the hypothesis that chronic pain alters CeA circuitry such that dynorphin neuron activity, normally neutral, becomes rewarding through KOR activation. Conversely, in the absence of pain, dynorphin release from these neurons to have minimal effect, although stimulation of these neurons in the presence of a KOR antagonist caused a place preference. This is not consistent with the plethora of studies that show the dynorphin/KOR system mediates aversive states 51 . Activation of the CeA dyn neurons may produce inhibition of local microcircuits or projections, though this is still unclear. The dynorphin/KOR signaling likely changes the output/input profiles within the CeA and to target brain regions. Acknowledgements We would like to acknowledge the following organizations for funding support: National Institute on Drug Abuse (Grant: 5R01DA053753 to C.M.C.), National Institute of General Medical Sciences (Grant: 2K12GM106996 to M.D.), National Institute on Mental Health (1ZICMH002963 to T.U.), the UCLA Carol Moss Spivak Scholars Fellowship and Phillip Foundation Fellowship to F.S., and the UCLA Shirley and Stefan Hatos Center for Neuropharmacology. We also acknowledge the UCLA Division of Laboratory Animal Medicine (DLAM) for their expert animal care and monitoring throughout the study. We thank Hoa Lam for her assistance with mouse colony management and laboratory organization. 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(n=6-10/gp, *P<0.05 unpaired t-tests, #P<0.05 one-sample t-test) E . Representative heat maps from postconditioning test day. F . No effect of CNO on mechanical sensitivity. (n=5-11/gp. Paired t-tests comparing baseline vs CNO. ns = not significant.). Pink dots indicate female mice; blue dots indicate male mice. CeA dyn : central amygdala dynorphin Figure 2. Activation of CeA dyn neurons is reinforcing in chronic pain state via the role of the kappa system. A . Experimental timeline of cross-over design for real time place preference (RTPP) with the kappa opioid receptor (KOR) antagonist aticrapant. B . Histological validation of channel rhodopsin (ChR) expression in CeA dyn neurons. C . Representative heat maps. D . Chronic constriction injury (CCI) mice have a significant preference for CeA dyn activation compared to pre-surgery. This effect is blocked by KOR antagonism. (n=18/gp. Two-way ANOVA Drug × Pain followed by Fisher’s post hoc comparisons. *P<0.05, ***P<0.001; ns = not significant.) E . Aticaprant has opposite effects on mice preference for the neural stimulation in sham and CCI groups (n=18/gp. Paired t-test. **P<0.01.) F . No effect of CeA dyn activation on mechanical sensitivity. (n=18/gp. Paired t-test. ns = not significant.). Pink dots indicate female mice; blue dots indicate male mice. CeA dyn : central amygdala dynorphin Figure 3. Whole CeA inhibition is not reinforcing but reduces mechanical sensitivity in chronic pain. A . Experimental timeline. B . Histological validation of Gi DREADD expression in the CeA. C . Time spent in vehicle- and clozapine N-oxide (CNO)-paired chamber for sham and chronic constriction injury (CCI) mice is not significantly different between groups. (n=10-11/gp. Two-way ANOVA Drug × Virus. ns = not significant.) D . Postconditioning scores show no effect of pain or virus between groups; CCI Gi DREADD mice have a significant preference for CNO. (n=15/gp. One sample t-tests. #P<0.05.) E . Mechanical withdrawal thresholds were not changed by CNO in sham or CCI mice of mcherry virally injected mice. CNO increased mechanical withdrawal thresholds in sham mice but reduced thresholds in CCI mice. *( n=4-7/gp. Paired t-tests baseline vs CNO. *P<0.05; ns = not significant.) CeA: central amygdala Figure 4. Projections of CeA dyn neurons. A . Whole brain image rotated to an angle that shows the injection site and major projections. Pseudo-coloring with an arbitrary scale is used to allow visualization of intense labeling at the injection site and relatively weak labeling in some projection areas. B-E . Cropped regions indicated by the dashed rectangles which were rotated to coronal orientation and then flattened as maximal intensity projections (B, 1mm; C, 0.84mm; D 1mm; E, 1.36mm). Non-linear intensity adjustments (gamma) were used to allow visualization of the intensity range and tissue background. Scale bars are 1mm. Note, dimensions refer to the imaged tissue, which is swollen by the tissue clearing process. Table 1. Statistical analyses. Figure 1 Figure 2 Figure 3 1 C Sham_mCherry, Sham_GiDREADD 8, 6 Two-way ANOVA Drug vs Virus Interaction F(1,11)=0.8058 P>0.05 Drug F(1,11)=0.2698 P>0.05 Virus F(1,11)=0.05176 P>0.05 CCI_mCherry, CCI_GiDREADD 6, 10 Two-way ANOVA Drug vs Virus Interaction F(1,13)=3.554 P>0.05 Drug F(1,13)=0.3382 P>0.05 Virus F(1,13)=5.523 P0.05 Virus F(1,24)=5.879 P<0.05 Pain F(1,24)=7.17 P<0.05 Sham_GiDREADD, CCI_GiDREADD 6,10 Unpaired t-test Pain - t(14)=2.904; P<0.05 CCI_mCherry, CCI_GiDREADD 6,10 Unpaired t-test Virus - t(14)=2.857; P0.05 Sham_GiDREADD 6 One-sample t-test for 0 - - P>0.05 CCI_mCherry 6 One-sample t-test for 0 - - P>0.05 CCI_GiDREADD 10 One-sample t-test for 0 - - P0.05 Sham_GiDREADD 5 Paired t-tests Baseline vs CNO - P>0.05 CCI_mCherry 7 Paired t-tests Baseline vs CNO - P>0.05 CCI_GiDREADD 11 Paired t-tests Baseline vs CNO - P>0.05 2 C Sham_mCherry 7 One-sample t-test for 0 - - P0.05 Sham_GiDREADD 100nl 4 One-sample t-test for 0 - - P>0.05 Sham_GiDREADD 150nl 18 One-sample t-test for 0 - - P>0.05 CCI_mCherry 7 One-sample t-test for 0 - - P>0.05 CCI_GiDREADD 75nl 7 One-sample t-test for 0 - - P>0.05 CCI_GiDREADD 100nl 3 One-sample t-test for 0 - - P<0.05 CCI_GiDREADD 150nl 18 One-sample t-test for 0 - - P<0.01 F Veh_Sham, Veh_CCI, Aticaprant_Sham, Aticaprant_CCI 18,18,18, 18 Two-way ANOVA Drug vs Pain Interaction F(1,17)=16.08 P0.05 Pain F(1,17)=2.285 P>0.05 Veh_Sham, Veh_CCI, 18 Comparisons (Fishers) Pain - P0.05 Veh_Sham, Aticaprant_Sham 18 Drug - P<0.05 Veh_CCI, Aticaprant_CCI 18 Drug - P<0.05 G Sham, CCI 18, 18 Paired t-test Pain - P0.05 3 C Sham_mCherry, Sham_GiDREADD 10 Two-way ANOVA Drug vs Virus Interaction F(1,9)=1.006 P>0.05 Drug F(1,9)=4.403 P>0.05 Virus F(1,9)= 5.885 P0.05 Drug F(1,10)=1.104 P>0.05 Virus F(1,10)=0.4235 P>0.05 D Sham_mCherry, Sham_GiDREADD, CCI_mCherry, CCI_GiDREADD 25 Two-way ANOVA Virus vs Pain Interaction F(1,19)=0.3058 P>0.05 Virus F(1,19)=0.9187 P>0.05 Pain F(1,19)=1.655 P>0.05 Sham_GiDREADD, CCI_GiDREADD 15 Unpaired t-test Pain - t(13)=1.700; P<0.05 CCI_mCherry, CCI_GiDREADD 15 Unpaired t-test Virus - t(10)=1.039; P0.05 Sham_GiDREADD 7 One-sample t-test for 0 - - P>0.05 CCI_mCherry 4 One-sample t-test for 0 - - P>0.05 CCI_GiDREADD 8 One-sample t-test for 0 - - P0.05 Sham_GiDREADD 7 Paired t-tests Baseline vs CNO - t(6)=3.562; P0.05 CCI_GiDREADD 7 Paired t-tests Baseline vs CNO - t(6)=2.553; P<0.05 Table 1 Information & Authors Information Version history V1 Version 1 06 February 2026 Copyright This work is licensed under a Non Exclusive No Reuse License. Authors Affiliations Merel Dagher University of California Los Angeles View all articles by this author Flora D’Oliveira da Silva University of California Los Angeles View all articles by this author Ted B. Usdin National Institute of Mental Health Intramural Research Program View all articles by this author Jamie E. Mondello University of California Los Angeles View all articles by this author Gabriella H. Sigal University of California Los Angeles Department of Integrative Biology & Physiology View all articles by this author nicolas massaly University of California Los Angeles Brain Research Institute View all articles by this author Catherine Cahill [email protected] University of California Los Angeles View all articles by this author Metrics & Citations Metrics Article Usage 267 views 111 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Merel Dagher, Flora D’Oliveira da Silva, Ted B. Usdin, et al. 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