Nerve injury triggers time-dependent activation of the locus coeruleus, influencing spontaneous pain-like behavior in rats.

Pain Medicine Anes hesiology, V 141 • NO 1 July 2024 131 ediTOR’S PeRSPecTiVe Wha We Al eady Know abou This Topic • Inc easing e idence sugges s he in ol emen o he no ad ene gic locus coe uleus in he dynamic modula ion o nocicep ion • The ole o neu onal p ojec ions om he locus coe uleus o he spi- nal co d and o he os al an e io cingula co ex in he modula ion o neu opa hic pain o e ime is incomple ely unde s ood Wha This A icle Tells us Tha Is New • In an expe imen al model o ch onic cons ic ion ne e inju y in a s, a combina ion o gene ic and his ologic app oaches e ealed a biphasic ime-dependen ole o locus coe uleus neu ons in mod- ula ing nocicep i e esponses • A e 2 days o ne e inju y, ac i a ion o locus coe uleus neu ons p ojec ing o spinal co d played a ole in a enua ing pain-like beha io while ac i a ion o locus coe uleus neu ons p ojec ing o he os al an e io cingula co ex ampli ied nocicep i e esponses • A e 30 days o ne e inju y, only he p ojec ions om locus coe- uleus neu ons o he os al an e io cingula co ex con ibu ed o he modula ion o pain-like beha io in his expe imen al model The locus coe uleus (LC) no ad ene gic sys em is he main sou ce o no ad enaline in he cen al ne ous sys em, and i is a key b ain a ea in ol ed in pain plas- ici y.1–3 Nume ous s udies indica e ha he LC is engaged by acu e noxious s imuli o in lamma ion, p omo ing eed- back inhibi ion o pain.4–6 Howe e , ecen s udies sugges ha he LC does no ul ill a uni o m ole in ch onic pain, bu a he , i changes dynamically h ough he ac i a ion o speci ic LC p ojec ions while o he s a e silenced as pain Ne e Inju y T igge s Time-dependen Ac i a ion o he Locus Coe uleus, In luencing Spon aneous Pain-like Beha io in Ra s I ene Suá ez-Pe ei a, Ph.D., Ca olina López-Ma ín, M.Sc., Ca men Cama ena-Delgado, Ph.D., Me i xell Llo ca-To alba, Ph.D., F ancisco González-Saiz, M.D., Rocío Ruiz, Ph.D., Ma iniano San iago, Ph.D., Es he Be ocoso, Ph.D. Anes hesiology 2024; 141:131–50 aBSTRacT Backg ound: Dynamic changes in neu onal ac i i y and in no ad ene gic locus coe uleus (LC) p ojec ions ha e been p oposed du ing he ansi ion om acu e o ch onic pain. Thus, he au ho s explo ed he cellula cFos ac i - i y o he LC and i s p ojec ions in conjunc ion wi h spon aneous pain-like beha io in neu opa hic a s. Me hods: Ty osine hyd oxylase:C e and wild- ype Long–E ans a s, males and emales, we e subjec ed o ch onic cons ic ion inju y (CCI) o 2 (sho - e m, CCI-ST) o 30 days (long- e m, CCI-LT), e alua ing cFos and Fluo o- Gold exp ession in he LC, and i s p ojec ions o he spinal co d (SC) and os al an e io cingula e co ex ( ACC). These es s we e ca ied ou unde basal condi ions (uns imula ed) and a e noxious mechanical s imula ion. LC ac i i y was e alua ed h ough chemogene ic and pha macologic app oaches, as we e i s p ojec ions, in associa ion wi h spon aneous pain-like beha io s. Resul s: CCI-ST enhanced basal cFos exp ession in he LC and in i s p o- jec ion o he SC, which inc eased u he a e noxious s imula ion. Simila basal ac i a ion was ound in he neu ons p ojec ing o he ACC, al hough his was no modi ied by s imula ion. S ong basal cFos exp ession was ound in CCI-LT, speci ically in he p ojec ion o he ACC, which was again no mod- i ied by s imula ion. No cFos exp ession was ound in he CCI-LT LCipsila e al (ipsi)/con ala e al (con a)→SC. Chemogene ics showed ha CCI-ST is associa ed wi h g ea e spon aneous pain-like beha io when he LCipsi is blocked, o by selec- i ely blocking he LCipsi→SC p ojec ion. Ac i a ion o he LCipsi o LCipsi/con a→ SC dampened pain-like beha io . Mo eo e , Designe Recep o Exclusi ely Ac i a ed by Designe D ugs (DREADDs)–media ed inac i a ion o he CCI-ST LCipsi→ ACC o CCI-LT LCipsi/con a→ ACC pa hway, o in a- ACC an agonism o α-ad eno ecep o s, also dampens pain-like beha io . conclusions: In he sho e m, ac i a ion o he LC a e CCI a enua es spon aneous pain-like beha io s ia p ojec ions o he SC while inc easing nocicep ion ia p ojec ions o he ACC. In he long e m, only he p ojec- ions om he LC o he ACC con ibu e o modula e pain-like beha io s in his model. (Anes hesiology 2024; 141:131–50) Supplemen al Digi al Con en is a ailable o his a icle. Di ec URL ci a ions appea in he p in ed ex and a e a ailable in bo h he HTML and PDF e sions o his a icle. Links o he digi al iles a e p o ided in he HTML ex o his a icle on he Jou nal’s Web si e (www.anes hesiology.o g). Pa o he wo k p esen ed in his a icle has been p esen ed as a pos e a In e na ional Associa ion o he S udy o Pain, To on o, Canada, Sep embe 19 h ough 23, 2022; and a Eu opean Pain Fede a ion, Dublin, I eland, Ap il 27 h ough 30, 2022. I.S.P. and C.L.M. con ibu ed equally o his a icle. Submi ed o publica ion July 14, 2023. Accep ed o publica ion Ma ch 26, 2024. Published online i s on Ap il 10, 2024. Copy igh © 2024 The Au ho (s). Published by Wol e s Kluwe Heal h, Inc., on behal o he Ame ican Socie y o Anes hesiologis s. This is an open-access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion-Non Comme cial-No De i a i es License 4.0 (CCBY-NC-ND), whe e i is pe missible o download and sha e he wo k p o ided i is p ope ly ci ed. The wo k canno be changed in any way o used comme cially wi hou pe mission om he jou nal. Anes hesiology 2024; 141:131–50. DOI: 10.1097/ALN.0000000000005006 The a icle p ocessing cha ge was unded h ough he au ho s’ ins i u ion. 132 Anes hesiology 2024; 141:131–50 Suá ez-Pe ei a e al. Pain Medicine ansi ions om acu e o ch onic, con ibu ing o bo h analgesia and pain.7 Indeed, 2 days a e ne e lesion, we ind s onge basal ac i i y in he LC ipsila e al o he side o lesion (LCipsi) han in he con ala e al LC (LCcon a), as wi nessed h ough he exp ession o cFos.8 S onge cFos exp ession was also e iden in he LCipsi neu ons p ojec - ing o he spinal co d (SC), and simila s ong cFos exp es- sion was e iden a e long- e m ne e damage (30 days). Howe e , in his la e case, he enhanced cFos exp ession was bila e al in he LC, and i was also e iden in neu ons p ojec ing o he os al an e io cingula e co ex ( ACC).8 The exp ession o cFos is a common ma ke o ecen cellula ac i i y, e lec ing cell ac i a ion induced by acu e e en s.9 Fo example, acu e s ess o nocicep i e s imulus inc eases cFos exp ession in he LC wi hin 1.5 o 2 h o he insul .10–13 The e o e, he physiologic signi icance o an inc ease in cFos 2 days o longe a e ne e inju y is in iguing. Fu he mo e, i is unclea whe he he c i ical ci cui s o he no mal p ocessing o acu e noxious inpu s a e dis up ed by ne e damage. I has been p oposed ha spon aneous s ong basal cFos exp ession migh possibly e lec he pa icipa ion o he LC in p ocessing spon aneous pain. Sensi i i y o e oked nocicep i e s imuli is enhanced in pa ien s expe iencing neu opa hic pain, as i is o spon aneous pain. Howe e , while pa oxysmal o ongoing spon aneous pain is consis- en ly iden i ied as a majo clinical complain (see e iew14), he unde lying mechanisms emain unclea . To u he explo e his issue, we assessed whe he cons ic ion damage o he scia ic ne e al e s he ac i i y o LC cells a ea ly and la e ime poin s a e lesion, bo h unde basal condi- ions (uns imula ed) and a e he applica ion o a noxious mechanical s imulus. Fu he mo e, as he LC is appa en ly composed o modules ha migh p oduce a ge ed neu o- modula ion,2,3,15 we assessed he ole o he LC as a whole, and bo h he LCipsi o LCcon a o he lesion. In addi ion, we assessed he empo al modula ion o pain by examin- ing he descending LC pa hway o he SC, as no ad enaline eleased in he SC supp esses pain-like beha io s.6,16 We also s udied he ACC, an a ea connec ed o he LC and impli- ca ed in neu opa hic pain.17 These s udies we e p omp ed by indings associa ing bila e al inc eases in no ad enaline in he p e on al co ex wi h long- e m neu opa hy,18 sugges - ing o e ac i a ion o he no ad ene gic sys em in p olonged pain condi ions. Consequen ly, cFos exp ession and Fluo o- Gold (FG; Fluo och ome, USA) s aining we e explo ed in he LC o Long–E ans a s subjec ed o ch onic cons ic- ion inju y (CCI) o 2 days (sho - e m, CCI-ST) o 30 days (long- e m, CCI-LT), analyzing how his esponded o noxious mechanical s imuli. Chemogene ics we e used o manipula e LC ac i i y and ha o he speci ic LC pa hways o he SC o ACC, pa icula ly in ela ion o spon aneous pain-like beha io s.8,18 In addi ion, pha macologic s ud- ies we e pe o med o e alua e he in ol emen o ACC ad ene gic ecep o s in spon aneous pain-like beha io s. Ma e ials and Me hods Animals T ansgenic a s we e ob ained by c ossing hemizygous y o- sine hyd oxylase–C e (TH:C e) ansgenic and wild- ype (WT) Long–E ans a s a he Uni e si y o Cádiz (Cádiz, Spain) om ounde s p o ided by he Ra Resou ce and Resea ch Cen e (Columbia, Missou i; dona ed by K. Deisse o h, Ph.D.), o WT Long–E ans a s p oduced in house o ob ained comme cially om Cha les Ri e (I aly) and om Jan ie Labo a o ies (F ance). Expe imen s we e ca ied ou on male TH:C e ansgenic o on male and emale WT Long–E ans a s (300 o 450 g), all main ained a he Uni e si y o Cadiz unde s anda d labo a o y con- di ions (22ºC, 12-h ligh /da k cycle, ligh s on 8:00 am, ood and wa e ad libi um). All animal handling and p oce- du es we e ca ied ou in acco dance wi h he Eu opean Commission (B ussels, Belgium) di ec i e (2010/63/ EU) and Spanish law (RD 53/2013) egula ing animal I ene Suá ez-Pe ei a, Ph.D.: Biomedical Resea ch Ne wo king Cen e o Men al Heal h (CIBERSAM), Ins i u e o Heal h Ca los III (ISCIII), Mad id, Spain; Neu opsychopha macology and Psychobiology Resea ch G oup, Depa men o Neu oscience, Facul y o Medicine, Uni e si y o Cádiz, Cádiz, Spain; Biomedical Resea ch and Inno a ion Ins i u e o Cádiz (INIBICA), Pue a del Ma Uni e si y Hospi al, Cádiz, Spain. Ca olina López-Ma ín, M.Sc.: Biomedical Resea ch Ne wo king Cen e o Men al Heal h (CIBERSAM), Ins i u e o Heal h Ca los III (ISCIII), Mad id, Spain; Neu opsychopha macology and Psychobiology Resea ch G oup, Depa men o Neu oscience, Facul y o Medicine, Uni e si y o Cádiz, Cádiz, Spain; Biomedical Resea ch and Inno a ion Ins i u e o Cádiz (INIBICA), Pue a del Ma Uni e si y Hospi al, Cádiz, Spain. Ca men Cama ena-Delgado, Ph.D.: Biomedical Resea ch and Inno a ion Ins i u e o Cádiz (INIBICA), Pue a del Ma Uni e si y Hospi al, Cádiz, Spain; IRCCS Humani as Resea ch Hospi al, Milan, I aly; Ins i u e o Neu oscience (IN-CNR), Na ional Resea ch Council o I aly, Milan, I aly. Me i xell Llo ca-To alba, Ph.D.: Biomedical Resea ch Ne wo king Cen e o Men al Heal h (CIBERSAM), Ins i u e o Heal h Ca los III (ISCIII), Mad id, Spain; Biomedical Resea ch and Inno a ion Ins i u e o Cádiz (INIBICA), Pue a del Ma Uni e si y Hospi al, Cádiz, Spain; Neu opsychopha macology and Psychobiology Resea ch G oup, Depa men o Cell Biology and His ology, Uni e si y o Cádiz, Cádiz, Spain. F ancisco González-Saiz, M.D.: Biomedical Resea ch Ne wo king Cen e o Men al Heal h (CIBERSAM), Ins i u e o Heal h Ca los III (ISCIII), Mad id, Spain; Depa men o Neu oscience, Facul y o Medicine, Uni e si y o Cádiz, Cádiz, Spain; Communi y Men al Heal h Uni o Villama in, Uni e si y Hospi al o Je ez de la F on e a, Cádiz, Spain. Rocío Ruiz, Ph.D.: Depa men o Biochemis y and Molecula Biology, Facul y o Pha macy, Ins i u e o Biomedicine o Se illa (IBiS) - Uni e si y Hospi al Vi gen del Rocío/CSIC/Uni e si y o Se illa, Se illa, Spain. Ma iniano San iago, Ph.D.: Depa men o Biochemis y and Molecula Biology, Facul y o Pha macy, Ins i u e o Biomedicine o Se illa (IBiS) - Uni e si y Hospi al Vi gen del Rocío/CSIC/Uni e si y o Se illa, Se illa, Spain. Es he Be ocoso, Ph.D.: Biomedical Resea ch Ne wo king Cen e o Men al Heal h (CIBERSAM), Ins i u e o Heal h Ca los III (ISCIII), Mad id, Spain; Neu opsychopha macology and Psychobiology Resea ch G oup, Depa men o Neu oscience, Facul y o Medicine, Uni e si y o Cádiz, Cádiz, Spain; Biomedical Resea ch and Inno a ion Ins i u e o Cádiz (INIBICA), Pue a del Ma Uni e si y Hospi al, Cádiz, Spain. Anes hesiology 2024; 141:131–50 133Suá ez-Pe ei a e al. Pain Changes Locus Coe uleus Ac i i y esea ch. Fu he mo e, all expe imen al p o ocols we e app o ed by he Commi ee o Animal Expe imen a ion a he Uni e si y o Cadiz. The s udy was conduc ed and he da a epo ed in acco dance wi h he Animal Resea ch: Repo ing o In Vi o Expe imen s (ARRIVE) guidelines.19 Neu opa hic Pain Model CCI o he scia ic ne e was used as a model o neu opa hic pain.20,21 Ra s we e anes he ized wi h iso lu ane (induc ion wi h 3 o 4% and main enance wi h 1.5 o 2.5%), and he le scia ic ne e was hen exposed a he mid- high le el, p oximal o he scia ic i u ca ion. Fou ch ome gu (4–0) liga u es we e ied loosely a ound he ne e, sepa a ed by 1.0 o 1.5 mm so as no o comp omise he ascula supply. The o e lying laye s o muscle we e hen closed wi h 4–0 non- abso bable silk h ead, and he skin was su u ed wi h 2–0 silk h ead. Sham ope a ions we e pe o med in he same man- ne bu wi hou ne e liga ion. The expe imen al p ocedu es we e ca ied ou 2 (ST) and 30 (LT) days a e CCI. Expe imen al Design and G oups All animals used in his s udy we e ini ially andomized in o g oups ha we e assigned di e en ea men s. In he expe - imen s in ol ing emale a s, hey we e ini ially assigned andomly o he di e en g oups, and subsequen ly, hei ho monal s a us was assessed by examining aginal smea s while hey we e unde anes hesia be o e applying noxious hind paw s imula ion.22 All s ages o he es ous cycle we e ep esen ed in each g oup, as de e mined by aginal cy ology. Be o e he beha io al expe imen s, all he animals unde wen ini ial aining and we e habi ua ed o bo h he expe imen al oom and handling. All he expe imen s and he analysis o he da a we e pe o med blind o he g oup and ea men assignmen s. Fu he mo e, powe analyses we e pe o med wi h G*Powe 23 (Hein ich-Heine-Uni e si ä Düsseldo , Düsseldo , Ge many) o de e mine he minimum sample size equi ed o he expe imen s in o de o de ec a signi i- can e ec using wo- ailed es s a an α le el = 0.05, powe = 0.80, and e ec size based on simila published s udies o cFos exp ession in he LC8 and paw linches in spon aneous pain es s.24 De ailed sample size ables o each igu e ( igs. 1 h ough 7) a e shown in Supplemen al Digi al Con en 2 (Supplemen al Tables 1 h ough 7, h ps://links.lww.com/ ALN/D532). Some expe imen al uni s o da a poin s we e excluded om he expe imen o s a is ical analysis i his o- logic e i ica ion indica ed e og ade ace injec ion o he si e o ca he e /cannula placemen was inaccu a e, o i he e was no Designe Recep o Exclusi ely Ac i a ed by Designe D ugs (DREADDs) exp ession in he LC. Fu he mo e, a G ubbs’s s a is ical es was employed o de ec ou lie s wi hin he da ase . These exclusion c i e ia we e p ede ined be o e conduc ing he analysis. Schemes ep esen ing he expe imen al p ocedu e a e shown in each igu e. In gene al, CCI was induced in male and emale adul Long–E ans a s a wo di e en ime poin s a e ne e inju y: 2 days (CCI-ST) and 30 days (CCI-LT). As con ol g oups, a s we e subjec ed o a sham su gical p ocedu e (sham g oup) o le unope a ed (nai e g oup). To explo e changes in LC ac i i y and i s p ojec ions o e ime, and wi h espec o noxious s imula ion, CCI and con- ol animals (sham o nai e) we e ini ially andomized in o he ollowing expe imen al g oups: S im(–), uns imula ed; o S im(+), s imula ed. Al e na i ely, o he chemogene ic modula ion o global no ad ene gic LC neu on ac i i y o o speci ic no ad ene gic LC p ojec ions, adeno-associa ed i us (AAV)–Gi–mChe y (DREADDs o chemogene ic inhibi ion), AAV-Gs-mChe y (DREADDs o chemo- gene ic ac i a ion), o AAV-mChe y (con ol i us) was injec ed in o he LCipsi and/o LCcon a o TH:C e a s. Clozapine-N-oxide (CNO, designe ligand) was adminis- e ed ia in ape i oneal injec ion, o locally (in a hecal o in a- ACC) ia mic oinjec ion in o he a ge si e 20 min be o e pe o ming he beha io al es s. In all hese expe i- men s, a g oup adminis e ed he ehicle alone was included as a con ol (saline [Sal] g oup). Finally, o he pha maco- logic blockade o ad eno ecep o s in he ACC, a cannula was implan ed bila e ally in o he ACC o all expe imen al a s, and he d ugs we e adminis e ed 30 o 20 min be o e pe o ming he beha io al es s (see sec ion “D ugs”). Simila ly, a g oup adminis e ed wi h he ehicle alone was included as a con ol (saline g oup) in hese expe imen s. Noxious Hind Paw S imula ion The hind paw o a s was s imula ed by applying pinched p essu e o he ipsila e al (le ) hind paw (600 g, 5 min) using a Analgesy-Me e (Ugo Basile, I aly), de ined as a mechan- ical noxious s imula ion, o enhance cFos exp ession. This s imulus was adminis e ed unde mild anes hesia wi h sodium pen oba bi al (50 mg/kg, in ape i oneally).26 Two hou s a e noxious s imula ion, all he a s we e anes he- ized deeply wi h an o e dose o sodium pen oba bi al, and hey ecei ed a ansca dial pe usion wi h saline, ollowed by 4% pa a o maldehyde (PFA) in 0.1 M phospha e bu e (PB), a e which hei SC and b ain we e emo ed.27 DREADDs App oaches DREADDs app oaches we e ca ied ou as indica ed p e i- ously, whe eby s ong colocaliza ion be ween mChe y and no ad ene gic neu ons was ound in he LC o TH:C e a s. Fo me hodologic alida ion o he DREADDs app oach, see Llo ca-To alba e al.,8,28 as well as Supplemen al Figu es 1 and 2 (Supplemen al Digi al Con en 1, h ps://links. lww.com/ALN/D531). S e eo axic Su ge y. The ollowing ec o s we e used in hese s udies: DREADDs (Vi us Vec o Co e, Gene The apy Cen e Vec o Co e a he Uni e si y o No h Ca olina, USA); AAV2/hSyn-DIO-hM4D(Gi)-mChe y 134 Anes hesiology 2024; 141:131–50 Suá ez-Pe ei a e al. Pain Medicine Fig. 1. S udy o locus coe uleus (lC) ac i a ion o e ime a e neu opa hy in uns imula ed and s imula ed male a s. (A) Scheme ep e- sen ing he expe imen al p ocedu e. The paw ipsila e al o he inju ed one was s imula ed in anes he ized a s by paw comp ession using an Analgesy-Me e (ugo Basile, I aly; 600 g, 5 min). The e ms ipsila e al and con ala e al a e ela i e o he ch onic cons ic ion inju y (CCI). (B) Rep esen a i e immunohis ochemis y o cFos labeled neu ons in he ipsila e al cen al lC (–9.72 o –9.96 om b egma25) in condi ions o no s imula ion (S im[–], black bo de s) o s imula ion (S im[+], ed bo de s) in nai e, sham–sho - e m (ST), sham–long- e m (lT), CCI-ST, and CCI-lT wild- ype a s (scale ba , 100 μm: cFos, ed; dopamine be a-hyd oxylase [DBH], g een). The whi e a ows illus a e examples o DBH-posi i e cells wi h cFos-posi i e nuclei in lC. (C) Expe imen al imeline o neu opa hy and ST (2 days) quan i ica ion o he cFos-posi i e neu ons in he cen al ipsila e al (I) and con ala e al (C) lC o nai e, sham-ST, and CCI-ST a s wi hou s imula ion (S im[–]) o a e applying a noxious s imulus (S im[+]). The mean + SD neu ons pe slice a e shown (each poin ep esen s an indi idual a ; n = 4–6 animals/g oup): **P < 0.01, ***P < 0.001 s. S im(–); +P < 0.05, ++P < 0.01, +++P < 0.001 s. lCipsi; #P < 0.05, ###P < 0.001 s. nai e o sham-ST ( h ee-way ANOVA wi h Tukey pos hoc es be ween nai e and sham-ST g oups, and be ween sham-ST and CCI-ST g oups). (D) Expe imen al imeline o neu opa hy and lT (30 days) quan i ica ion o he cFos-posi i e neu ons in he cen al ipsila e al (I) and con ala e al (C) lC o sham-lT and CCI-lT a s wi hou s imula ion (S im[–]) o wi h noxious s imula ion (S im[+]). The mean + SD neu ons pe slice a e shown (each poin ep esen s an indi idual a ; n = 6 animals/g oup): ***P < 0.001 s. S im(–); ##P < 0.01, ###P < 0.001 s. sham-lT ( h ee-way ANOVA wi h Tukey pos hoc es ). Anes hesiology 2024; 141:131–50 135Suá ez-Pe ei a e al. Pain Changes Locus Coe uleus Ac i i y Fig. 2. S udy o he ac i a ion o he locus coe uleus (lC)→spinal co d (SC) pa hway o e ime a e neu opa hy in uns imula ed and s im- ula ed male a s. (A) Scheme o he e og ade Fluo o-Gold (FG; Fluo och ome, uSA) ace s a egy and ep esen a i e images (scale ba , 500 μm) o FG adminis a ion si e and lC neu ons ha p ojec o he ipsila e al spinal co d (SC) in wild- ype a s. Dis ibu ion o FG labeling in ipsila e al (ipsi) and con ala e al (con a) lC neu ons (mean + SD o he numbe o neu ons pe slice; each poin ep esen s an indi idual a ; n = 4 animals: S uden ’s es ). (B) Rep esen a i e immuno luo escence o he cen al lC (–9.72 o –9.96 om b egma25) showing he DBH, cFos, and FG exp ession, as well as a me ged image. The whi e a ows illus a e examples o cFos-posi i e cells, and he inse shows an example o cFos/FG-labeled DBH-posi i e neu ons (scale ba , 100 μm: DBH, g een; cFos, ed; FG, yellow). (C) Expe imen al imeline o neu opa hy and sho - e m (ST, 2 days) quan i ica ion o he cFos/FG labeled neu ons in he cen al ipsila e al (I) and con ala e al (C) lC o sham-ST and CCI-ST a s wi hou s imula ion (S im[–]) o wi h noxious s imula ion (S im[+]). The mean + SD neu ons pe slice a e shown (each poin ep esen s an indi idual a ; n = 6 animals/g oup): *P < 0.05, **P < 0.01 s. S im(–); +P < 0.05, ++P < 0.01 s. lCipsi; #P < 0.05 s. sham-ST ( h ee-way ANOVA wi h Tukey pos hoc es ). (D) Expe imen al imeline o neu opa hy and long- e m (lT, 30 days) quan i ica ion o he cFos/FG-labeled neu ons in he cen al ipsila e al (I) and con ala e al (C) lC o sham-lT and CCI-lT a s wi hou s imula ion (S im[–]) o wi h noxious s imula ion (S im[+]). The mean + SD neu ons pe slice a e shown (each poin ep esen s an indi idual a ; n = 6 animals/g oup): non-signi ican di e ences ( h ee-way ANOVA wi h Tukey pos hoc es ). CCI, ch onic cons ic ion inju y; DBH, dopamine be a-hyd oxylase; 4V, ou h en icle. 136 Anes hesiology 2024; 141:131–50 Suá ez-Pe ei a e al. Pain Medicine Fig. 3. S udy o he ac i a ion o he locus coe uleus (lC)→ os al an e io cingula e co ex ( ACC) pa hway o e ime a e neu opa hy in uns imula ed and s imula ed male a s. (A) Ca oon o he e og ade Fluo o-Gold (FG; Fluo och ome, uSA) ace s a egy a ge ing lC neu- ons ha p ojec o he bila e al ACC and ep esen a i e images (scale ba , 500 μm) o FG adminis a ion si e and lC neu ons ha p ojec bila e ally o he ACC in wild- ype a s. The dis ibu ion o FG label in he ipsila e al (ipsi) and con ala e al (con a) lC neu ons was e alua ed (mean + SD o he numbe o neu ons pe slice; each poin ep esen s an indi idual a ; n = 4 animals: S uden ’s es ). (B) Rep esen a i e immuno luo escence o he cen al lC (–9.72 o –9.96 om b egma25) showing he DBH, cFos, and FG exp ession, as well as a me ged image. The whi e a ows illus a e examples o cFos-posi i e cells, and he inse shows an example o cFos/FG-labeled DBH-posi i e neu ons (scale ba , 100 μm: DBH, g een; cFos, ed; FG, yellow). (C) Expe imen al imeline o neu opa hy and sho - e m (ST, 2 days) quan i ica ion o he cFos/FG-labeled neu ons o he cen al ipsila e al (I) and con ala e al (C) lC o sham-ST and CCI-ST a s wi hou s imula ion (S im[–]) o a e applying a noxious s imulus (S im[+]). The mean + SD neu ons pe slice a e shown (each poin ep esen s an indi idual a ; n = 5 animals/ g oup): **P < 0.01, ***P < 0.001 s. (S im[–]); ++P < 0.01, +++P < 0.001 s. lCipsi; ##P < 0.01, ###P < 0.001 s. sham-ST ( h ee-way ANOVA wi h Tukey pos hoc es ). (D) Expe imen al imeline o neu opa hy and long- e m (lT, 30 days) quan i ica ion o he cFos/FG-labeled neu ons o he cen al ipsila e al (I) and con ala e al (C) lC o sham-lT and CCI-lT a s wi hou s imula ion (S im[–]) o wi h noxious s imu- la ion (S im[+]). The mean + SD neu ons pe slice a e shown (each poin ep esen s an indi idual a ; n = 5 o 6 animals/g oup): **P < 0.01, ***P < 0.001 s. (S im[–]); +++P < 0.001 s. lCipsi; ##P < 0.01, ###P < 0.001 s. sham-lT ( h ee-way ANOVA wi h Tukey pos hoc es ). CCI, ch onic cons ic ion inju y; DBH, dopamine be a-hyd oxylase; 4V, ou h en icle. Anes hesiology 2024; 141:131–50 137Suá ez-Pe ei a e al. Pain Changes Locus Coe uleus Ac i i y (hM4D[Gi]-DREADD) inhibi o i us, i e 3 × 1012 g/ ml, 1.4 µl/ LC; AAV2/hSyn-DIO- M3D(Gs)-mChe y ( M3D[Gs]-DREADD) ac i a o i us, i e 3 × 1012 g/ ml, 1.4 µl/LC; and AAV2/hSyn-DIO-mChe y (con ol- DREADD), i e 5.6 × 1012 g/ml, 1.4 µl/LC. This con ol ec o con ained a mChe y epo e p o ein wi hou he DREADD epo e . DREADDs ec o s we e injec ed in o he LC o TH:C e a s anes he ized (in ape i oneally) wi h ke amine (100 mg/kg, Rich e Pha ma, Spain) and xylazine (20 mg/ Fig. 4. E ec o chemogene ic inhibi ion and ac i a ion o he ipsila e al and con ala e al locus coe uleus (lC) on spon aneous pain-like beha io a di e en imes a e ne e inju y. (A) Scheme o he no ad ene gic lC inhibi ion o ac i a ion using he DREADD s a egy in TH:C e male a s and ep esen a i e immuno luo escence image (scale ba , 10 μm) o mChe y exp ession in he no ad ene gic lC neu ons (DBH, g een; mChe y, ed). (B) Expe imen al imeline o neu opa hy and quan i ica ion o he sho - e m (ST, 2 days) and long- e m (lT, 30 days) ipsila e al paw linches o sham and CCI a s in he spon aneous pain es a e ipsila e al lC (lCipsi) o con ala e al lC (lCcon a) no ad ene gic inhibi ion by CNO (1 mg/kg in ape i oneal, mean + SD, each poin ep esen s an indi idual a ; n = 4–6 animals/g oup: *P < 0.05, ***P < 0.001, s. sham-Sal; ++P < 0.01 s. CCI-Sal, h ee-way epea ed measu es ANOVA wi h Tukey pos hoc es ). (C) Expe imen al imeline o neu opa hy and long- e m (lT, 30 days) quan i ica ion o he ipsila e al paw linches o sham and CCI a s in he spon aneous pain es a e ipsila e al lC (lCipsi) o con ala e al lC (lCcon a) no ad ene gic ac i a ion by CNO (1 mg/kg in ape i oneal, mean + SD, each poin ep esen s an indi idual a ; n = 8 o 9 animals/g oup: ***P < 0.001 s. sham-Sal; +++P < 0.001 s. CCI-Sal, K uskal–Wallis es ollowed by Mann– Whi ney u es ). AAV, adeno-associa ed i us; CCI, ch onic cons ic ion inju y; CNO, clozapine N-oxide; DBH, dopamine be a-hyd oxylase; DREADD, Designe Recep o Exclusi ely Ac i a ed by Designe D ug; Sal, saline; SP, spon aneous pain-like es ; TH, y osine hyd oxylase. 138 Anes hesiology 2024; 141:131–50 Suá ez-Pe ei a e al. Pain Medicine Fig. 5. E ec o chemogene ic inhibi ion and ac i a ion o ipsila e al and con ala e al locus coe uleus (lC)→spinal co d (SC) pa hway on spon aneous pain-like beha io o e ime a e ne e inju y. (A) Expe imen al imeline o lCipsi-SC o lCcon a-SC pa hway inhibi ion a sho - e m (ST, 2 days) and long- e m (lT, 30 days) a e neu opa hy using he DREADD s a egy in male TH:C e a s. Quan i ica ion o he ipsila e al paw linches o sham and CCI a s in he spon aneous pain-like es a e ipsila e al lC (lCipsi) o con ala e al lC (lCcon a) no ad ene gic inhibi ion by CNO (3 μM in a hecal, mean + SD, each poin ep esen s an indi idual a ; n = 4 animals/g oup: *P < 0.05, ***P < 0.001, s. sham-Sal; +P < 0.05 s. CCI-Sal, h ee-way epea ed measu es ANOVA wi h Tukey pos hoc es ). (B) Expe imen al imeline o lCipsi→SC o lCcon a→SC pa hway ac i a ion in he sho - e m (ST, 2 days) and long- e m (lT, 30 days) a e neu opa hy using he DREADD s a egy in TH:C e a s. Quan i ica ion o he ipsila e al paw linches o sham and CCI a s in he spon aneous pain es a e ipsila e al lC (lCipsi) o con- ala e al lC (lCcon a) no ad ene gic ac i a ion by CNO (3 μM in a hecal, mean + SD, each poin ep esen s an indi idual a ; n = 3–7 animals/ g oup: **P < 0.01, ***P < 0.001 s. sham-Sal; ++P < 0.01, +++P < 0.001 s. CCI-Sal, h ee-way epea ed measu es ANOVA wi h (Con inued ) Anes hesiology 2024; 141:131–50 139Suá ez-Pe ei a e al. Pain Changes Locus Coe uleus Ac i i y kg, Calie Labo a o y, S.A., Spain), which we e placed in o a s e eo axic ame wi h he skull le el along bo h he an e o- pos e io (AP) and medio-la e al (ML) axis o p ecise a ge ing. C anial windows we e opened abo e he LC coo dina es (AP, –3.2 mm; ML, ±1.3 mm; do so- en al [DV], 6.2 mm), wi h he head o ien ed a a 15-deg ee angle o he ho izon al plane. A e injec ion, he wound was cleaned wi h 0.9% saline and disin ec ed wi h iodine poly- idone, and he skin was su u ed wi h 4-0 nonabso bable silk h ead. To enhance ec o exp ession, he beha io al s udies we e ca ied ou 3 weeks a e he injec ions. Vi al Exp ession. Animals we e pe used wi h PFA (4%) a he end o he expe imen s, and hei b ains we e col- lec ed and p ocessed28,29 o e i y he i al injec ion si e and he exp ession o mChe y in he LC by immuno lu- o escence. All he LC sec ions (30 µm) we e incuba ed o 48 h a 4ºC wi h an an ibody agains ed luo escen p o ein (1:500 [5F8], Ch omo ek, Ge many) and mouse an idopa- mine be a hyd oxylase (DBH, 1:1,000, Me ck Chemicals & Li e Science S.A., Spain). Subsequen ly, he an ibodies we e de ec ed wi h a bio inyla ed donkey an i a an ibody (1:200, Jackson ImmunoResea ch Eu ope, Uni ed Kingdom), which was isualized wi h Alexa Fluo 568 s ep a idin o a don- key an imouse Alexa Fluo 488 (1:1,000, In i ogen, USA). The sec ions we e hen washed and co e slipped in luo o-gel aqueous moun ing medium, and he images we e acqui ed on a Zeiss LSM 900 Con ocal mic oscope wi h Ai yscan 2 (Ca l Zeiss Mic oscopy GmbH, Ge many). The selec i e exp es- sion o DREADD was also assessed in he A5 no ad ene gic nucleus and en al egmen al a ea, he la e p obed wi h a abbi an i-TH p ima y an ibody (1:1,000) ha was isualized wi h a donkey an i abbi Alexa Fluo 488 (In i ogen, USA). T ace App oach FG was used as a e og ade ace (4%/0.4 µl) o he ACC o SC. Fo FG injec ions a he lumba le el o he SC,8 a s we e anes he ized wi h sodium pen oba bi al (50 mg/ kg, in ape i onally, Ve oquinol S.A., Spain), and local anal- gesia was adminis e ed subcu aneously wi h ca p ophen (10 mg/kg, Zoe is, Spain) and bupi acaine (2 mg/kg, B. B aun Medical S.A., Spain). Addi ional doses o pen oba - bi al we e adminis e ed as necessa y. Ra s we e placed in a s e eo axic ame, and a laminec omy was pe o med a he le el o he T12–L2 e eb ae o expose he L4–L6 seg- men s o he SC. The du a was hen emo ed, and FG was adminis e ed a ou si es along he ipsila e al L4–L6 do sal ho n o he SC (SCipsi). A e FG injec ion, he skin was closed wi h s aples, and he wound was cleaned wi h 0.9% saline and disin ec ed wi h iodine poly idone. In ano he se o expe imen s, he FG was mic oinjec ed unila e al o bila e ally in o he ACC acco ding o he same s e eo axic su ge y pe o med in he DREADD expe imen s (see sec- ion “DREADDs App oaches”).8 The a ge coo dina es o ACC we e AP + 3.0 mm, ML ± 1.0 mm, and DV 1.2 mm, wi h he head o ien ed a a 0-deg ee angle o he ho izon- al plane. To enhance ace exp ession, he neu oana omical s udies we e ca ied ou 4 days a e he injec ions, and he loca ion o he FG injec ion si e was e i ied o be wi hin he SC and ACC in all he animals s udied. Sequen ial SC and ACC (40 µm) co onal sec ions we e isualized di ec ly on an Olympus BX60 luo escence mic oscope equipped wi h an Olympus DP74 came a (Spain). A e e i ica ion, he animals in which he FG injec ion was no app op ia ely loca ed in he a ge a ea we e excluded om he analysis. ACC Cannula Implan a ion Fo in a- ACC d ug adminis a ion, cannulae we e implan ed by s e eo axic su ge y.8 The a s we e anes he- ized wi h an in ape i oneal injec ion o ke amine (100 mg/ kg) and xylazine (20 mg/kg), and hen placed in a s e eo- axic ame. The cannulae we e implan ed bila e ally in o he ACC (AP + 3.0 mm, ML ± 1.0 mm, and DV 1.2 mm) wi h he head o ien ed a a 0º angle o he ho izon al plane. The cannulae we e ixed in place wi h den al cemen and ou ancho sc ews, and main ained closed by inse ing a s ainless s eel wi e un il he es session. A e cannula implan a ion, he a s we e allowed o eco e o 1 week be o e he beha io al es s began. To e i y cannula place- men , 0.5 μl Pon amine Sky Blue (Sigma–Ald ich, Spain) was injec ed in o he in a- ACC jus be o e he animals we e sac i iced. Sequen ial co onal sec ions (40 µm) o he ACC we e ul ima ely ob ained on a Sliding Mic o ome (Mic om HM 450, Fishe Scien i ic SL, Spain) coupled o BFS-MP eezing s age (Physi emp Ins umen s, USA). A e e i ica ion, he animals in which he cannula was no app op ia ely loca ed in he a ge a ea we e excluded om he analysis (Supplemen al Figu es 3, 4, and 5, Supplemen al Digi al Con en 1, h ps://links.lww.com/ALN/D531). In a hecal Ca he e Ins alla ion Fo in a hecal d ug injec ion, a PE-10 ca he e (0.61 mm diame e ) was implan ed in o he lumba SC unde iso- lu ane anes hesia.30 An incision was made in he skin, and a cannula (20-gauge, 0.9 × 40 mm) was in oduced Fig. 5. (Con inued) Tukey pos hoc es ). (C) Expe imen al imeline o neu opa hy and long- e m (lT, 30 days) quan i ica ion o he ipsila e al paw linches o sham and CCI a s in he spon aneous pain es a e clonidine (20 μg in a hecal) adminis a ion (mean + SD, each poin ep esen s an indi idual a ; n = 5 o 6 animals/g oup: **P < 0.01 s. sham-Sal; ++P < 0.01 s. CCI-Sal, K uskal–Wallis es ollowed by Mann–Whi ney u es ). AAV, adeno-associa ed i us; CCI, ch onic cons ic ion inju y; Cloni, clonidine; CNO, clozapine N-oxide; DREADD, Designe Recep o Exclusi ely Ac i a ed by Designe D ug; Sal, saline; SP, spon aneous pain; TH, y osine hyd oxylase. 146 Anes hesiology 2024; 141:131–50 Suá ez-Pe ei a e al. Pain Medicine SC, which enhances no ad enaline elease and in u n con- ibu es o he blockade o ascending nocicep i e inpu s.6,8,16 Ano he ele an pa hway is ha o he LC→ ACC, as lesion47,48 o injec ion o a b ain-de i ed neu o ophic ac- o (BDNF)– opomyosin ecep o kinase B (T kB) an ag- onis in o he ACC comple ely blocks he condi ioned place p e e ence induced by he α-2 ad eno ecep o ago- nis Cloni.48,49 Hence, he ACC appea s o be necessa y o egula e spon aneous pain. In CCI-ST animals, he e is also a s ong and la e alized ac i i y in his egion, such ha chemogene ic blockade o he LCipsi→ ACC dampens spon aneous pain, ye no e ec is ound when he LCcon a is manipula ed. O e all, his sugges s ha la e alized cFos exp ession is ela ed o spon aneous pain-like beha io , albei in opposing di ec ions depending on he p ojec ion. In long- e m neu opa hy, la e alized cFos exp ession is los , and while s ong exp ession is ound in he LC glob- ally,8,50 his is no he case o cells p ojec ing o he SC. DREADDs-media ed blockade o ei he he LCipsi o he LCcon a globally o o he speci ic p ojec ion o he SC does no modi y spon aneous pain-like beha io s. This esponse is consis en wi h he belie ha he LC does no induce endogenous analgesia h ough he descending p ojec ion o he SC when neu opa hy becomes long- e m.8,51 The e is also a obus bila e al inc ease in cFos a he ACC le el in CCI-LT, and he e is a educ ion in spon aneous pain- like beha io s when he LCipsi/con a is blocked. Hence, he LC→ ACC p ojec ion appea s o ul ill a p onocicep i e ole in bo h sho - and long- e m pain. This aligns wi h p e- ious indings showing ha chemogene ic ac i a ion o he LC→P e on al co ex p ojec ion exace ba es spon aneous pain, p oduces a e sion, and inc eases anxie y-like beha io in neu opa hic pain animals.16 On he o he hand, when he LC is chemogene ically ac i a ed globally, o simply i s p o- jec ion o he SC, he e is signi ican elie o pain- ela ed beha io s.8 These chemogene ic s udies a e consis en wi h he pha macologic e ec s o he acu e in a hecal admin- is a ion o he selec i e no ad ene gic eup ake inhibi o eboxe ine, which elie es e oked pain and induces con- di ioned place p e e ence. Howe e , acu e sys emic ebox- e ine adminis a ion also elie es e oked pain, al hough i is a e si e in he condi ioned place p e e ence pa adigm.52 Hence, he bene icial e ec o no ad enaline a he SC is p obably coun e ac ed by he ac i a ion o o he sup aspinal no ad ene gic p ojec ions. Ou da a also sugges ha he hype ac i i y o he LC→ ACC p ojec ion ha induces spon aneous pain-like beha io is media ed by ac i a ion o α-1 ad eno ecep o s o e he sho e m, and by α-1 and α-2 ad eno ecep o s in he long e m. P e ious indings showed mic oinjec ion o he α-2 ad eno ecep o s agonis Cloni in o he ACC alle ia es spon aneous pain-like beha io a day 7 bu no a 14 days a e ne e inju y in common pe oneal ne e– liga ed mice.48 These da a, in a di e en animal model, sugges an analgesic e ec o ACC α-2 ad eno ecep o ac i a ion, which con as s wi h ou indings, al hough i is consis en wi h he dynamic egula ion o cingula e α- ad eno ecep o s o e ime a e ne e inju y. We also explo ed cFos le els a e mechanical noxious s imula ion o a hind paw. In nai e animals, cFos was only weakly exp essed, bu i inc eased bila e ally and uni o mly in he LC when noxious s imula ion was applied o one hind paw, consis en wi h p e ious da a.11,13 Sham animals beha e like nai e animals in he long e m, which may indi- ca e sa is ac o y esolu ion o he damage p oduced by he sham in e en ion. Findings om nai e and sham-LT ani- mals sugges ha bila e al ac i a ion o he LC du ing acu e unila e al hind paw pain could pa ly e lec he modula ion o nocicep i e p ocessing in he SC do sal ho n. Howe e , his is unlikely because his pa hway is no endogenously ac i a ed when FG/cFos colabeling o neu ons is explo ed in sham-LT animals. Fu he mo e, chemogene ic inhibi ion o LC-no ad ene gic neu ons o in a-LC lidocaine admin- is a ion did no modi y senso ial esponses in sham-LT animals.2,8,24,53 Ano he possibili y is ha his bila e al LC ac i a ion is ela ed o o he nonsenso ial aspec s o pain, such as he inhe en s ess associa ed wi h he pain expe i- ence i sel .7,54 In his sense, es ain s ess inc eases anxie y beha io and cFos immuno eac i i y in LC neu ons, and he chemogene ic inhibi ion o LC-no ad ene gic neu ons p e en s s ess-induced anxie y.40 The e o e, LC ac i i y appea s o ha e a mino in luence on e oked hype sensi i - i y in hese condi ions, which could be ela ed o a s ess- ela ed esponse. In sham-ST animals, he la e alized basal cFos exp es- sion is u he enhanced by noxious s imula ion o he hind paw. In e es ingly, simila da a we e ob ained om he LCipsi o CCI-ST a s, al hough cFos exp ession was signi ican ly s onge , sugges ing ha he LC esponses a his poin o neu opa hy esemble o he o ms o sho - e m inju y, ye hey a e mo e in ense. Fu he mo e, cFos exp ession in he CCI-ST LCcon a did no di e signi ican ly om ha in he LCcon a o sham animals, and i did no change upon nox- ious s imula ion, pe haps sugges ing ha he LCcon a had al eady eached i s maximum possible esponse. These da a a e consis en wi h he signi ican inc ease in hype sensi i - i y o a cold s imulus in CCI-ST animals a e chemogene ic inhibi ion o he LCipsi.8 Fu he mo e, we p e iously ound ha pha macologic blockade wi h lidocaine signi ican ly inc eased he nocicep i e h eshold o Sp ague–Dawley a s.24 As no e ec was ound when blocking he LCcon a,8,24 endogenous LC-d i en analgesia in he sho e m unde e oked condi ions appea s o be media ed h ough he LCipsi. A e y di e en p o ile is ound in he long e m a e neu opa hy, whe eby he baseline inc ease in cFos exp es- sion is no al e ed by noxious s imuli, ei he globally in he LC o in he speci ic p ojec ions o he SC o ACC. This is consis en wi h ea lie da a showing ha 11 days o ne e inju y a enua ed es ain -induced global LC cFos exp ession,55 sugges ing ha long- e m ne e inju y migh Anes hesiology 2024; 141:131–50 147Suá ez-Pe ei a e al. Pain Changes Locus Coe uleus Ac i i y impai he o dina y cellula esponse o he LC o an acu e s imulus. This is again in line wi h educed LC-media ed descending inhibi o y con ols o diminished analgesic e icacy o d ugs a ge ing LC pa hways desc ibed p e i- ously.56–60 Acco dingly, chemogene ic blockade o he LCipsi o LCcon a a CCI-LT does no modi y he esponse o ace- one,8 sugges ing ha his inc ease in cFos is no ela ed o he senso y dimension o pain. Howe e , blockade o he LCcon a wi h lidocaine ele a es he nocicep i e h eshold in CCI-LT Sp ague–Dawley a s.24 Fu he mo e, enhanced LC elec ophysiologic esponses a e p oduced by mechan- ical s imula ion o he ne e-inju ed paw a CCI-LT, p e- dominan ly media ed by he LCcon a.61 These indings align wi h he esponses o Sp ague–Dawley a s o bila e al mic oinjec ions o lidocaine in o he LC, which comple ely e e se he beha io al signs o neu opa hy 2 weeks a e ne e inju y.62 O e all, he LC seems o ul ill dis inc oles depending on he a s ain, wi h a la e alized p onocicep- i e ac i i y o he LC associa ed wi h long- e m neu op- a hy in Sp ague–Dawley bu no Long–E ans a s. These disc epancies may e lec he epo ed s ain di e ences in he no ad ene gic pa hways o hese animals.63–66 The new da a p esen ed he e sugges ha bila e al LC o LC→SC p ojec ions ul ill an analgesic ole in spon aneous and e oked condi ions, mainly due o he ac i a ion o he LCipsi, al hough ac i a ion o he LCipsi→ ACC pa hway pa - ially coun e ac s endogenous analgesia in esponse o spon- aneous pain. In he long e m, cFos is exp essed s ongly on a global le el in esponse o pain, and in he ACC, ye i is no longe la e alized and he e is no ac i a ion in he SC. Impo an ly, cFos exp ession is no modi ied by noci- cep i e s imula ion a e long- e m pain. A his ime poin , he analgesic con ibu ion o he LC is los , and he ac i i y o he LCipsi→ ACC pa hway in p omo ing spon aneous pain p edomina es. An icipa ing o hcoming elec ophysiologic s udies o complemen hese expe imen s, speci ic LC p o- jec ions o he SC o ACC appea o ha e di e en beha - io al e ec s depending on he ime ha has passed since he inju y. Mo eo e , o he p ojec ion a eas like he do sal e icu- la nucleus24 o insula co ex67 could also po en ially con ib- u e o he spon aneous pain media ed by he LC and wa an u he explo a ion. Fu u e s udies u ilizing he a ge ed ecombina ion o ac i e popula ions (TRAP) app oach68–70 could p o ide insigh s as o whe he dis inc subpopula ions wi hin he LC a e ec ui ed o spon aneous pain, and help elucida e hei physiologic and molecula cha ac e is ics. Finally, al hough ou s udy did no e eal di e ences be ween male and emale a s, i is c ucial o include bo h sexes in such esea ch o comp ehensi ely unde s and any sexual dimo - phism o common beha io s ela ed o hese phenomena. Acknowledgmen s The au ho s a e e y g a e ul o Jose An onio Ga cia Pa ida, M.Sc., Uni e si y o Cádiz (Cádiz, Spain), and Elena Ma ín Ál a ez, Highe Technician, Uni e si y o Cádiz (Cádiz, Spain) o hei excellen echni- cal assis ance. They also acknowledge he assis ance o he Cen al Se ices o Scien i ic and Technological Resea ch, Heal h Sciences and Animal Resea ch a he Uni e si y o Cádiz. Resea ch Suppo This s udy was suppo ed by g an s PID2022-142785OB-I00 and PDC2022-133987-100 unded by MCIN/AEI/ 10.13039/501100011033 and, as app op ia e, by “ERDF A way o making Eu ope,” by he Eu opean Union and Eu opean Union Nex Gene a ionEU/PRTR espec i ely, by Andalusian Regional Minis y o Heal h (Spain) (PI- 0134-2018; No. P20-00958), by he Regional Ope a ional P og am o Andalucía FEDER, by he Biomedical Resea ch and Inno a ion Ins i u e o Cádiz (INIBICA, Spain) (IN- C09), by he Andalusian Regional Minis y o Economy, Inno a ion, Science and Employmen (Spain) (CTS-510), and by he Biomedical Resea ch Ne wo king Cen e o Men al Heal h (CIBERSAM, Spain) (CB07/09/0033). The igu es we e gene a ed wi h BioRende .com (BioRende , Canada). Compe ing In e es s The au ho s decla e no compe ing in e es s. Co espondence Add ess co espondence o D . Be ocoso: Neu opsychopha macology and Psychobiology Resea ch G oup, Depa men o Neu oscience, Facul y o Medicine, Uni e si y o Cádiz, 11003 Cádiz, Spain. es he .be [email p o ec ed] Supplemen al Digi al Con en Supplemen al Digi al Con en 1: Supplemen al Figu es, h ps://links.lww.com/ALN/D531 Supplemen al Digi al Con en 2: Sample Size Tables 1 h ough 7, h ps://links.lww.com/ALN/D532 Supplemen al Digi al Con en 3: S a is ical Tables 8 h ough 14, h ps://links.lww.com/ALN/D533 Supplemen al Digi al Con en 4: Table 15: HPLC Resul s, h ps://links.lww.com/ALN/D534 Re e ences 1. Llo ca-To alba M, Bo ges G, Ne o F, Mico JA, Be ocoso E: No ad ene gic locus coe uleus pa hways in pain modula ion. Neu oscience 2016; 338:93–113 2. Schwa z LA, Luo L: O ganiza ion o he locus coe uleus-no epineph ine sys em. Cu Biol 2015; 25:R1051–6 148 Anes hesiology 2024; 141:131–50 Suá ez-Pe ei a e al. Pain Medicine 3. Poe GR, Foo e S, Eschenko O, e al.: Locus coe uleus: A new look a he blue spo . 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