Parabrachial bombesin receptor subtype 3 neurons facilitate heat pain in persistent inflammation

Parabrachial bombesin receptor subtype 3 neurons facilitate heat pain in persistent inflammation | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Parabrachial bombesin receptor subtype 3 neurons facilitate heat pain in persistent inflammation View ORCID Profile Heather N. Allen , View ORCID Profile Tyler S. Nelson , Nia A. Dufeal , Naomi K. Grabus , Kai Trevett , Jessica Merrett , View ORCID Profile Rajesh Khanna doi: https://doi.org/10.1101/2025.03.05.641630 Heather N. Allen 1 Department of Pharmacology & Therapeutics, McKnight Brain Institute, College of Medicine, University of Florida , Gainesville, FL, USA 2 Center for Advance Pain Therapeutics and Research (CAPToR), University of Florida , Gainesville, FL, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Heather N. Allen Tyler S. Nelson 1 Department of Pharmacology & Therapeutics, McKnight Brain Institute, College of Medicine, University of Florida , Gainesville, FL, USA 2 Center for Advance Pain Therapeutics and Research (CAPToR), University of Florida , Gainesville, FL, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Tyler S. Nelson Nia A. Dufeal 1 Department of Pharmacology & Therapeutics, McKnight Brain Institute, College of Medicine, University of Florida , Gainesville, FL, USA 2 Center for Advance Pain Therapeutics and Research (CAPToR), University of Florida , Gainesville, FL, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Naomi K. Grabus 1 Department of Pharmacology & Therapeutics, McKnight Brain Institute, College of Medicine, University of Florida , Gainesville, FL, USA 2 Center for Advance Pain Therapeutics and Research (CAPToR), University of Florida , Gainesville, FL, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Kai Trevett 3 Department of Molecular Pathobiology, College of Dentistry, New York University , New York, NY, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jessica Merrett 3 Department of Molecular Pathobiology, College of Dentistry, New York University , New York, NY, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Rajesh Khanna 1 Department of Pharmacology & Therapeutics, McKnight Brain Institute, College of Medicine, University of Florida , Gainesville, FL, USA 2 Center for Advance Pain Therapeutics and Research (CAPToR), University of Florida , Gainesville, FL, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Rajesh Khanna For correspondence: r.khanna{at}ufl.edu Abstract Full Text Info/History Metrics Preview PDF Abstract The parabrachial nucleus (PBN) is a critical hub for pain processing that acts as a switchboard for nociceptive signals, relaying them to forebrain regions that integrate sensory signals with affective aspects of pain. Despite strong evidence for the PBN in pain modulation, the heterogeneity of parabrachial neurons poses a challenge in defining their specific contributions. Here, we identify bombesin receptor subtype 3 ( Brs3 )-expressing neurons as a distinct glutamatergic PBN subpopulation involved in inflammatory heat pain. Using Fos expression analysis and in vivo calcium imaging, we demonstrate that Brs3 neurons exhibit heightened activity in response to noxious and innocuous stimuli following an inflammatory insult. Chemogenetic activation of Brs3 neurons in uninjured mice induces pain-like behaviors, indicating their sufficiency in driving nociceptive responses. While inhibition of Brs3 neurons does not reverse inflammatory pain-induced mechanical allodynia, it effectively reduces heat hypersensitivity, suggesting a specific role in thermal pain processing. Brs3 -expressing neurons encompass multiple previously identified pain-related PBN subpopulations, including those expressing the mu opioid receptor ( Oprm1 ), tachykinin 1 receptor ( Tacr1 ), and neuropeptide Y Y1 receptor ( Npy1r ), positioning Brs3 as a potential unifying marker of heat hypersensitivity circuits. These findings provide new insight into the organization of pain-processing networks in the PBN and highlight Brs3 neurons as a crucial population for inflammatory heat pain. Introduction Pain is a multifaceted experience and essential protective mechanism that helps the body maintain homeostasis and avoid injury 1 . The Internation Association for the Study of Pain’s most recent definition of pain emphasizes that “pain cannot be inferred solely from activity in sensory neurons”, acknowledging the necessity of supraspinal circuits in creating and shaping the aversive nature of the pain experience 2 . Although pain originates in the periphery, its perception depends on intricate supraspinal processing, where emotional and cognitive inputs are integrated with physical nociceptive signals to ultimately produce the unpleasant sensation of pain 3 . Inflammatory pain, the result of tissue injury and immune activation, is a hallmark of many chronic pain conditions 4 , including arthritis 5 , post-surgical pain 6 , low back pain 7 , fibromyalgia 8 , nerve damage 9 , endometriosis 10 , and inflammatory bowel disease 11 . Persistent inflammatory pain can lead to prolonged hypersensitivity and maladaptive changes to nociceptive circuits, contributing to the transition from acute to chronic pain 12 , 13 . A deeper understanding of how inflammatory pain alters pain circuits is critical for designing effective therapeutics. The parabrachial nucleus (PBN) is a hindbrain hub highly involved in pain processing, relaying peripheral nociceptive signals via the spinal cord to forebrain regions, including the amygdala, extended amygdala, thalamus, and hypothalamus 14 . In chronic pain conditions, PBN neurons exhibit heightened activity in response to noxious stimuli, contributing to the amplification of pain observed in both neuropathic 15 – 20 and inflammatory 20 – 22 pain conditions. Glutamatergic neurons, which comprise approximately 85% of all PBN neurons 23 , are particularly important contributors to pain modulation: they are activated by inflammatory pain conditions 24 – 26 , and their activation alone is sufficient to induce hypersensitivity in mice, even in the absence of injury 16 . However, glutamatergic neurons in the PBN are highly heterogenous, and there are dozens of distinct subpopulations defined by expression of various neuropeptides and receptors 23 . Several distinct glutamatergic subpopulations have been directly implicated in pain processing. PBN neurons expressing the mu opioid receptor 27 , tachykinin 1 receptor 28 , or neuropeptide Y Y1 receptor-expressing neurons 29 have each separately been shown to contribute to the modulation of inflammatory pain in mice. However, while some co-expression exists between these populations 23 , 29 , 30 , they exhibit significant anatomical and molecular divergence, making it challenging to determine how they synchronize to modulate pain in the PBN. Using recent spatial transcriptomic studies 23 , we have identified a promising candidate for unifying these diverse pain-modulating populations in the PBN: neurons expressing the transcript for bombesin receptor subtype 3 ( Brs3 ). Brs-3, classified as a bombesin-related receptor based on homology, is an orphan Gq coupled receptor in mammals with no known endogenous ligand 31 . Unlike other bombesin-related receptors, Brs-3 does not bind gastrin releasing peptide, or neuromedin B 31 , making its functional role elusive. However, Brs3 expression in the PBN marks a distinct subset of neurons that encompasses the separate mu opioid receptor-, tachykinin 1 receptor-, and neuropeptide y y1 receptor-expressing PBN populations 23 . This unique molecular identity positions Brs3 -expressing neurons as a potential key integrator of inflammatory pain processing in the PBN. To test the hypothesis that Brs3 -expressing neurons contribute to the modulation of inflammatory pain, we utilized molecular, genetic, and behavioral approaches to probe the parabrachial Brs3 population in a mouse model of persistent inflammation. Methods Animals Male and female C57Bl/6 (Jackson labs, #000664) and Brs3 Cre mice (B6.Cg- Brs3 tm1.1(cre/GFP)Rpa/J ) 32 aged 6-10 weeks were used for all experiments. Animals were group housed in a temperature and humidity controlled room on a 12:12 hour light:dark cycle with ad libitum access to food and water. All procedures were approved by the Institutional Animal Care and Use Committee at the University of Florida (IACUC protocol #202400000002). All experiments include both male and female animals. Surgery Male and female Brs3 Cre ( Brs3 Cre , Brs3 Cre/+ and Brs3 Cre/Cre ) and Brs3 +/+ wildtype mice were anesthetized using isoflurane (5% induction, 2.5% maintenance) and had their heads fixed in a stereotax and a small incision made in the scalp. For fiber photometry experiments, a craniotomy was performed over the right PBN (coordinates A/P −5.15 mm, M/L +/− 1.1 mm, D/V −3.35 mm) and 200 nL of a Cre-dependent virus carrying a genetically encoded calcium indictor (AAV9-hSyn-flex-GCamp6s, Addgene, #100845) was injected at a rate of 1 nL/sec using a Nanoject III programmable nanoliter injector (Drummond Scientific) connected to a glass pipette. The injector was left in place for an additional five minutes to prevent backflow and to allow the virus to diffuse. One bone screw was attached to the skull left of midline anterior to lambda and one bone screw was attached right of midline anterior to bregma. A fiberoptic implant (RWD; 1.25 mm ferrule diameter, 200 mm core, 0.37 NA) was implanted above the injection site in the right PBN and secured to the skull using dental cement. For chemogenetic experiments, bilateral craniotomies were performed over the left and right PBN and 200 nL of Cre-dependent virus carrying an excitatory (AAV8-hSyn-DIO-hM3Dq-mCherry, Addgene, #44361), inhibitory (AAV8-hSyn-DIO-hM4Di-mCherry, Addgene, #44362), or control (AAV8-hSyn-DIO-mCherry, Addgene, #50459) construct was injected at a rate of 1 nL/sec. For permanent genetic silencing experiments, 200nL of a virus carrying Cre-dependent diphtheria toxin A (AAV8-Ef1a-mCherry-flex-dtA, Canadian Neurophotonics Platform Viral Vector Core Facility) was injected into the left and right PBN of Brs3 Cre and and Brs3 +/+ animals at a rate of 1 nL/sec. The injector was left in place for an additional five minutes to prevent backflow and to allow the virus to diffuse. The scalp was closed using 6.0 silk suture and triple antibiotic ointment was applied to the wound. Animals received a subcutaneous injection of Meloxicam (20 mg/kg, Patterson Veterinary Supply), and recovered on a heating pad before being returned to their homecage, where they received acetaminophen water (1.1 mg/kg) for 72 hours post surgery. Mice were allowed at least three weeks of recovery post brain surgery before undergoing behavior experiments. Drugs Under 2% isoflurane anesthesia, 20 mL of undiluted Complete Freund’s Adjuvant (CFA, Millipore Sigma, #AR001) was injected into the plantar surface of the left hindpaw using a 30 gauge needle attached to a 25 mL Hamilton syringe. Control animals were anesthetized with 2% isoflurane but received no injection in the hindpaw. Compound 21 (Hello Bio, #HB6124) was diluted in saline the day of the experiment and injected intraperitoneally (1 mg/kg) 15 minutes (spontaneous behavior) of 30 minutes (reflexive behavior) before behavioral testing. Behavior All behavior was performed blinded to drug injection (C21 or saline) and/or virus (hM4Di, hM3Dq, mCherry). All experiments include both male and female mice. Fiber photometry Fiber photometry experiments were conducted as previously described 17 , 18 , 33 , 34 . Animals were habituated to Plexiglas chambers, elevated wire mesh, and patch cable attachment for 1 hour each two days prior to experimentation. On the day of experimentation, animals were habituated in Plexiglas chambers on elevated wire mesh for one hour before having patch cables attached to the implanted fiberoptics and habituating for an additional 30 minutes with the experimenter in the room. Baseline recordings were performed to innocuous and noxious stimuli according to the following paradigm: 0.07 g von Frey filament, 1.0 g von Frey filament, blunted pin prick. Calcium transients were collected continuously (FP3002, Neurophotometrics) during the stimulation protocol. Each stimulus was applied to the plantar surface of the left hindpaw three times two minutes apart, and all three responses were averaged to represent the animal’s response to that stimulus. Custom MatLab scripts were used to normalize the GCamp6s (470 nm) signal to the isobestic signal (405 nm), controlling for motion artifacts as well as photobleaching. The change in fluorescence (dF/F) was calculated by subtracting the GCamp6s signal during stimulation from the average GCamp6s signal over the 10 seconds directly prior to stimulation. The area under the curve (AUC) was calculated for the five seconds directly following stimulus application. Following baseline recordings, animals received a CFA injection in the left hindpaw, and the fiber photometry protocol was repeated 3 days after CFA. All animals were perfused following the experiment for histological verification of viral injection and fiber optic placement in the PBN. Spontaneous pain behavior Mice were habituated to the experimental room for 1-2 hours prior to experimentation. Animals received an intraperitoneal injection (1 mg/kg) of compound 21 (C21) and were returned to their homecage for 15 minutes. Animals were then placed in a clear Plexiglas chamber surrounded by mirrors, and a video camera recorded the animal for the 15 minutes. Spontaneous pain behavior was defined as agitated licking, scratching, biting, and paying attention to a specific area. Behavior videos were quantified (seconds spent displaying agitated pain behavior) by two separate experimenters and their counts were averaged for each animal. Mechanical sensitivity Mechanical withdrawal threshold were determined using the von Frey assay as previously described 35 . Mice were habituated to Plexiglas chambers on an elevated wire mesh platform for 1.5 hours prior to testing. Calibrated von Frey filaments (Braintree Scientific, #58011) were used to assess mechanical sensitivity at the plantar surface of the left hindpaw using the up/down method 36 . Cold withdrawal latency Directly following mechanical sensitivity testing, mice were assessed for cold sensitivity using the acetone droplet test 35 . Using a 5 mL syringe attached to PE-90 tubing with a flared end, approximately 10 mL of acetone was applied to the plantar surface of the left hindpaw. The time in seconds the animal spent licking, biting, and attending to the paw was measured. The test was repeated three times and the average of all three trials is reported. Heat withdrawal latency Thermal thresholds were assessed using the hotplate assay as previously described 35 . Mice were gently placed on a 52°C Ugo Basile hot/cold plate within an acrylic enclosure (Stoelting #55075). The time until the hindpaw withdrawal response, defined as jumping, licking, flinching, was recorded in seconds. The animal was immediately removed after withdrawal or a cutoff of 30 seconds to avoid tissue damage. Three trials were averaged with a between-trial interval of at least 10 minutes. Fluorescence in situ hybridization Three days after CFA, RNAscope multiplex fluorescent V2 assay was used with probes for Brs3 (ACD Bio #454111-C3) and Fos (ACD Bio #316921). Brains were extracted and post-fixed in 10% neutral buffered formalin for 2 hours at 4°C before being transferred to 30% sucrose and stored at 4°C for 3 days. Tissue was embedded in optimal cutting temperature gel (OCT, TissueTek) before being sectioned at 20 mm on a cryostat. Three to five representative PBN sections from each animal were mounted on SuperFrost Plus microscope slides (Fisher Scientific) and allowed to airdry overnight at room temperature. Slides were rinsed in MilliQ water for five minutes to remove OCT followed by two consecutive baths of 100% ethanol (2 minutes each). Tissue was treated with Protease III for 20 minutes in HybEZ oven at 40°C. RNAscope was performed according to manufacturer’s instructions (ACD Bio Inc) as previously described 37 . Briefly, tissue was incubated with probes for Brs3 and Fos for two hours at 40°C before undergoing a series of amplification steps according to manufacturer’s instructions (AMP1 30 min, AMP2 30 min, AMP 3 15 min) at 40°C. Each probe had a TSA-based fluorescent label developed in succession followed by application of DAPI for 30 seconds. Slides were coverslipped with VECTASHIELD hardset anti-fade mounting medium with DAPI (Vector Laboratories, #H-1400-10) and imaged on a Leica DMI8 microscope (Wetzlar, Germany) using a 20x objective lens. Images were analyzed using QuPath software v0.4.3. The lateral PBN was outlined according to the mouse reference brain atlas (Allen Institute) and cells with greater than three puncta ( Fos, Brs3 ) surrounding a DAPI labeled nucleus were considered positive. Cell were considered positive for co-expression if they had greater than three puncta for each marker. Between 3 and 5 sections from across the rostral-caudal axis of the PBN were quantified and averaged for each animal. Immunohistochemistry Animals receiving viral injections were transcardially perfused following behavioral experiments with ice cold PBS followed by ice cold 10% neutral buffered formalin. Brains were extracted and post fixed in 10% neutral buffered formalin overnight at 4°C. Brains were then transferred to 30% sucrose at 4°C for five days before being frozen and sectioned at 35 mm on a cryostat. Floating PBN sections were washed 3 times for 5 minutes each in PBS before being incubated in blocking buffer (5% normal goat serum, 0.1% Triton X-100 in PBS) for sixty minutes. Sections were then incubated in a 1:1000 dilution of primary antibody against either GFP (Rb anti-GFP, Millipore Sigma, #AB3080) for fiber photometry animals or mCherry (rabbit anti-mCherry, Abcam, #ab167453) for chemogenetic and dtA animals overnight. Following three 5 minute washes, sections were incubated in secondary antibody (1:1000 goat anti rabbit Alexa Fluor 488, Invitrogen, #A11008, or 1:1000 goat and rabbit Alexa Fluor 594, Invitrogen, #A21442) for 90 minutes. After three 5 minute washes in 1% phosphate buffer, sections were mounted on SuperFrost Plus microscope slides and coverslipped with VECTSHIELD hardset mounting medium with DAPI (Vector Laboratories, #H-1400-10). Images were captured on Echo Revolution microscope using a 20x objective lens. Viral infection was confirmed by comparing fluorescently labeled cells to the Allen Brain atlas to align with the PBN. Statistics and data analysis Data were analyzed using GraphPad Prism v10.4.1 and all values are presented as mean ± SEM. Statistical significance is determined as P<0.05. Fiber photometry data, hotplate, spontaneous behavior, and in situ co-expression data were analyzed using two tailed paired and unpaired Student’s T-tests, as appropriate. Mechanical sensitivity and dtA hotplate data were analyzed using two-way analysis of variance (ANOVA) followed by Tukey’s post hoc and Uncorrected Fisher’s LSD for multiple comparisons. Results Neurons in the PBN exhibit hyperexcitability during inflammatory pain 20 , 24 , 25 . To investigate whether Brs3 -expressing neurons are activated by persistent inflammatory pain, we used in situ hybridization to visualize co-expression of Brs3 with Fos mRNA in the PBN of control and CFA animals ( Figure 1A-H ). Animals with inflammatory pain induced by CFA displayed a larger number of Fos positive Brs3 cells in the PBN compared to control animals, suggesting that Brs3 PBN neurons are indeed activated by CFA ( Figure 1I ). Download figure Open in new tab Figure 1: Co-expression of Brs3 with Fos in the PBN of animals following CFA-induced inflammatory pain. (A) Representative PBN (white dashed line) labeled for Brs3 and Fos expression in a control animal. (B) Inset of Fos expression in the PBN of a control animal. (C) Inset (yellow box) of Brs3 expression in PBN of a control animal. (D) Merged inset of Fos and Brs3 co-expressed in the PBN of a control animal. (E) Representative PBN labeled for Brs3 and Fos expression in a CFA animal. (F) Inset of Fos expression in the PBN of a CFA animal. (G) Inset of Brs3 expression in PBN of a CFA animal. (H) Merged inset of Fos and Brs3 co-expressed in the PBN of a CFA animal. (I) Quantified co-expression of the number of PBN cells expressing both Brs3 and Fos (unpaired t-test, p=0.0006, n=3-4 animals, average of 3-5 sections per animal). Next, we used in vivo fiber photometry to evaluate calcium activity of Brs3 PBN neurons during stimulation of the hindpaw both before and after induction of inflammatory pain with CFA ( Figure 2A-2C ). During baseline recordings, Brs3 neurons in the PBN were unresponsive to innocuous ( Figure 2D, 2H ) mechanical stimulation but slightly responsive to noxious ( Figure 2L ) mechanical stimulation. However, 3 days after CFA-induced inflammatory pain, calcium responses in Brs3 PBN neurons increased to both innocuous ( Figure 2E-2G, 2I-2K ) and noxious ( Figure 2M-2O ) stimulation compared to baseline. These results complement our molecular findings ( Figure 1 ) and demonstrate that Brs3 neurons are more responsive to mechanical stimulation after inflammatory pain. Download figure Open in new tab Figure 2: CFA increases activity of Brs3 PBN neurons. (A) Schematic of viral and fiberoptic implant surgery in the PBN. (B) Representative viral expression of GCamp6 in Brs3 neurons within the PBN (region demarcated by white dashed line). (C) Timeline schematic for the fiber photometry experiments. Averaged calcium transients in response to 0.07 g von Frey filament at baseline (D) and after CFA (E). (F) Heat map showing averaged responses across animals at baseline and after CFA in response to a 0.07 g filament. (G) Area under the curve summary data for the five seconds directly following stimulation with a 0.07 g filament (paired t-test, p=0.0026, n=13). Averaged calcium transients in response to 1.0 g von Frey filament at baseline (H) and after CFA (I). (J) Heat map showing averaged responses across animals at baseline and after CFA in response to a 1.0 g filament. (K) Area under the curve summary data for the five seconds directly following stimulation with a 1.0 g filament (paired t-test, p=0.0086, n=13). Averaged calcium transients in response to blunted pin prick at baseline (L) and after CFA (M). (F) Heat map showing averaged responses across animals at baseline and after CFA in response to a blunted pin prick. (G) Area under the curve summary data for the five seconds directly following stimulation with a blunted pin prick (paired t-test, p=0.0162, n=13). Solid black line indicates time of stimulus application. Solid trace represents average response, shaded region represents SEM. To evaluate if increased activity of Brs3 PBN neurons influences pain behavior, we used cell-type specific excitatory chemogenetics in Brs3 Cre mice ( Figure 3A-3B ). Chemogenetic activation of Brs3 PBN neurons with compound 21 induced mechanical allodynia in animals transfected with the excitatory designer receptor exclusively activated by designer drug (DREADD) hM3Dq, indicated by reduced withdrawal thresholds compared to baseline ( Figure 3C ). Similarly, C21-induced activation of PBN Brs3 neurons also produced profound cold allodynia, as evidenced by increased withdrawal durations during the acetone droplet assay ( Figure 3D ). C21 had no effect in mCherry transfected control animals ( Figure 3C-3D ). Activation of Brs3 neurons in the PBN also induced heat allodynia, demonstrated via decrease withdrawal latency on a 52°C hotplate compared to mCherry control animals ( Figure 3E ). Finally, chemogenetic activation of Brs3 PBN neurons with C21 provoked spontaneous nocifensive behavior, measured via time spent licking, biting, and actively attending to the back and paw ( Figure 3F ). Together, these results indicate that activation of Brs3 neurons in the PBN is sufficient to produce pain-like behaviors in uninjured animals. Download figure Open in new tab Figure 3: Activation of Brs3 -expressing PBN neurons produces pain-like behaviors. (A) Viral schematic for infecting Brs3 PBN neurons with Cre-dependent excitatory chemogenetic (hM3Dq) or control (mCherry) virus. (B) Representative viral expression in the PBN (regions demarcated by white dashed line). (C) Mechanical withdrawal thresholds in animals infected with control mCherry or excitatory hM3Dq virus at baseline and after intraperitoneal injection of saline or compound 21 (C21) (two-way ANOVA, effect of interaction p<0.0001, with Tukey’s multiple comparisons, n=12). (D) Withdrawal duration to the acetone droplet assay in animals infected with mCherry control or excitatory hM3Dq virus a baseline and after intraperitoneal injection of saline or C21 (two-way ANOVA, effect of interaction p<0.0001, with Tukey’s multiple comparisons, n=12). (E) Withdrawal latency on a 52°C hotplate in control and hM3Dq animals after intraperitoneal injection with C21 (unpaired t-test, p=0.006, n=12). (F) Time animals infected with hM3Dq spent exhibiting spontaneous pain-like behaviors after intraperitoneal injection of saline or C21 (paired t-test, p=0.0301, n=12). Finally, we tested the necessity of Brs3 neurons in inflammation-induced pain-like behavior. Using Cre-dependent inhibitory chemogenetics ( Figure 4A ), we tested the effect of to transiently inhibiting Brs3 PBN neural activity on pain-like behaviors associated with CFA-induced persistent inflammation. Although CFA produced mechanical allodynia in animals transfected with hM4Di, transient inhibition of PBN Brs3 neurons with C21 was unable to completely reverse mechanical allodynia. Interestingly, temporary inhibition of Brs3 PBN neurons was sufficient to reverse heat allodynia, as C21-mediated inhibition increased animals’ latency to withdrawal on a 52°C hotplate after CFA ( Figure 4C ). Download figure Open in new tab Figure 4: Brs3-expressing PBN neurons are important for heat but not mechanical hypersensitivity. (A) Schematic for infecting Brs3 PBN neurons with Cre-dependent inhibitory chemogenetic (hM4Di) virus. (B) Mechanical withdrawal thresholds before and after CFA in response to intraperitoneal injection of saline or C21 (Two-way ANOVA, effect of time p=0.6788, effect of CFA p<0.0001, effect of interaction p=0.1648, n=10). (C) Withdrawal latency on a 52°C hotplate before and after inhibition of PBN Brs3 neurons in hM4Di animals treated with CFA (Unpaired t-test, p=0.0002, n=10). (D) Schematic for permanent genetic silencing of Brs3 -expressing neurons in the PBN. (E) Mechanical withdrawal thresholds before and after CFA in Brs3 Cre/+ and Brs3 +/+ mice (Two-way ANOVA, effect of treatment p<0.0001, effect of genotype p=0.0996, effect of interaction p=0.1474, n=9). (F) Withdrawal latency on a 52°C hotplate before and after CFA in animals with intact PBN Brs3 and animals with ablated PBN Brs3 (Two-way ANOVA, effect of interaction p=0.0021, Uncorrected Fisher’s LSD, n=9). We also tested the hypothesis that permanent genetic deletion of Brs3 PBN neurons would prevent the development of CFA-induced pain-like behaviors. Cre-dependent diphtheria toxin A (DTA) was injected into the bilateral PBN of Brs3 Cre and Brs3 +/+ mice 3 weeks prior to behavioral testing ( Figure 4D ). Genetic deletion of PBN Brs3 had no effect on baseline behavior or on the development of mechanical allodynia following CFA ( Figure 4E-4F ). However, parabrachial Brs3 -ablated mice failed to develop heat hypersensitivity after CFA, in contrast to wildtype controls ( Figure 4F ). Together, this data suggests that Brs3 PBN neurons contribute to the development of CFA-induced heat but not mechanical hypersensitivity. Discussion Recent years have seen a resurgence of interest in the role of the parabrachial nucleus (PBN) in pain processing 15 , 16 , 21 , 23 – 25 , 28 , 29 , 33 , 34 , 37 – 42 . Advancements in technology have enabled rapid experimental progress in dissecting the PBN’s contribution to pain modulation since it was originally identified as a vital node in the pain pathway in the late 1980s 43 , 44 . New studies have highlighted not only the PBN’s critical involvement in pain but also its remarkable heterogeneity 16 , 23 . Here, we have utilized data produced from an elegant spatial transcriptomic study of the parabrachial nucleus in pain 23 to identify Brs3 -expressing neurons as a distinct and functionally relevant PBN subpopulation involved in modulating heat hypersensitivity associated with inflammatory pain. We demonstrate for the first time that Brs3 neurons in the PBN play a role in pain modulation. Parabrachial Brs3 -expressing neurons exhibit increased Fos expression in response to complete Freund’s adjuvant (CFA)-induced inflammation and heightened calcium transients in response to both innocuous and noxious mechanical stimuli following CFA treatment. Together, these results suggest that parabrachial Brs3 -expressing neurons are activated by inflammatory pain. Interestingly, Brs3 neurons were largely unresponsive to mechanical stimulation before the onset of pain (baseline), suggesting that their recruitment occurs only after inflammatory processes enhance excitatory input to the PBN. Despite this recruitment, our results indicate that Brs3 neurons are not necessary for CFA-induced mechanical allodynia, as neither transient nor permanent inhibition of these neurons reversed mechanical hypersensitivity. This suggests that Brs3 neurons are likely one of several PBN subpopulations contributing to inflammatory pain-related mechanical allodynia, reflecting the broader increase in activation of PBN neurons observed following injury. Glutamatergic neurons in the PBN are activated by inflammatory pain 20 , 24 , 25 , but the precise subpopulations contributing to specific types of pain are still being discovered. Glutamatergic PBN neurons expressing the mu opioid receptor or the tachykinin 1 receptor are important for inflammatory pain, as inhibition of either of these populations reduces pain-like behaviors induced by the formalin assay 27 , 28 . Neuropeptide Y Y1 receptor-expressing parabrachial neurons are also activated by inflammatory pain, and inhibition or ablation of these neurons reverses inflammatory pain-like behaviors 29 . However, these studies focus on spontaneous pain-like behaviors or mechanical sensitivity as experimental readouts for inflammatory pain; none of them assess heat hypersensitivity, a hallmark of inflammatory pain conditions 45 . While inhibition of parabrachial Brs3 neurons was unable to completely reverse inflammatory pain-induced mechanical allodynia, we found them to be critically involved in modulating heat hypersensitivity. Brs3 -expressing PBN neurons represent a unique subset of glutamatergic neurons that encapsulate established neural subpopulations already implicated in inflammatory pain - Oprm1 , Tacr1 , and Npy1 - and may serve as a key integrative hub that drives heat hyperalgesia in the context of inflammatory pain. Beyond the parabrachial nucleus, Brs3 -expressing PBN neurons project to the hypothalamus, thalamus, lateral preoptic area, and amygdala, suggesting their potential involvement in integrating nociceptive signals with affective and homeostatic processing 32 . Pain is intricately involved in homeostasis-it reflects an adverse condition that affects autonomic functions and causes a behavioral response 1 . Given that terminal regions Brs3 -expressing PBN neurons project to also regulate energy metabolism, emotion, and negative affect, it is likely that parabrachial Brs3 neurons have additional physiological roles beyond pain modulation 32 . Parabrachial projections to different brain regions are to be involved in discrete aspects of the pain experience 40 . Thus, future studies should investigate whether downstream parabrachial Brs3 projections contribute to the emotional and aversive aspects of pain as well as autonomic functions affected by pain conditions. Limitations and Future Directions One limitation of this study is the inability to accurately measure calcium transients in Brs3 neurons in response to heat stimulation. Although it is possible to observe calcium responses during heat stimulation, technical challenges prevent precise time-locking of heat withdrawal responses to calcium recordings, as heat-induced reflexes have an inherent latency that complicates stimulus alignment. Future improvements in experimental design may help address this issue. Since CFA-induced inflammation does not produce cold allodynia, we did not assess Brs3 neuron involvement in cold sensitivity. However, our data reveal that activating Brs3 neurons increases hypersensitivity in the acetone droplet test, raising the possibility that Brs3 neurons contribute to cold allodynia in conditions such as neuropathic pain, where cold hypersensitivity is a prominent feature 46 . Other persistent pain conditions with temperature related allodynia should be explored. Additionally, since both the PBN and Brs3 -expressing neurons are involved in various physiological processes, it will be critical to assess potential off-target effects by monitoring vital signs and other readouts of homeostatic behavior (e.g. feeding) during Brs3 neuron stimulation. Similarly, the contribution of Brs3 PBN neurons to non-reflexive measures of pain, such as aversion-based behavioral assays, could also clarify whether Brs3 neurons influence the affective dimensions of pain. Conclusion The PBN is a highly complex hub that processes and relays a vast array of sensory inputs, including pain. There is growing interest in dissecting the contributions of specific excitatory subpopulations within the PBN to distinct aspects of the pain experience. Our findings suggest that different PBN populations process inflammation-related mechanical and thermal hypersensitivity separately, supporting the idea that pain is not a monolithic experience but instead consists of multiple parallel pathways. Disclosures R. Khanna is the co-founder of Regulonix LLC, a company that develops non-opioid drugs for the treatment of chronic pain. No other authors have declared conflicts of interest. Author contributions HNA, TSN, and RK developed the concept. HNA designed and conducted the experiments, completed the data analysis, and wrote the manuscript. TSN helped conduct in situ and behavior experiments. NAD and NKG assisted with tissue processing and conducted immunohistochemistry. KT and JM quantified behavioral videos. All authors had the opportunity to discuss the results and comment on the paper. Acknowledgements We thank Dr. Richard Palmiter for generously donating the original Brs3 Cre mice. BioRender.com was used for making schematics. 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