A molecularly defined basalo-prefrontal-thalamic circuit regulates sensory and affective dimensions of pain in male mice

Domain: neuroscience
Published: 2026-01-29
Editorial outcome: major_revision

Abstract
Boththemedialprefrontalcortex(mPFC)andthalamushavebeenimplicated
inpainregulation.However,therolesofthemPFC-thalamusconnectionin
painandhowthemPFCmodulatesnociceptiveprocessingremainunclear.
Here,weshowthatthemPFCneuronsprojectingtothalamus,markedby
Foxp2expression,aredeactivatedinbothacuteandchronicpaininmalemice.
PersistentinactivationofthemPFCFoxp2+neuronsenhancesnociceptive
sensitivity,whiletheiractivationalleviatesmultipleaspectsofpain.Circuit-
specificmanipulationsrevealedthattheprojectionstoparataenialnucleus,
mediodorsalandventromedialthalamusdifferentiallymodulatesensoryand
affectivepain.Additionally,themPFCFoxp2+neuronsreceivecholinergic
inputfromthebasalforebrain,particularlythehorizontaldiagonalband
(HDB).Notably,activationoftheα4β2-containingnicotinicacetylcholine
receptorinmPFCexertsantinociceptiveeffectsinFoxp2+neuron-dependent
manner.Together,ourstudydefinesanHDB→mPFCFoxp2→thalamuscircuit
essentialforsensoryandaffectivepainmodulationandunderscoresthe
therapeuticpotentialoftargetingmPFCcholinergicsignalinginchronicpain
management.

Introduction
Pain is an unpleasant sensory and emotional experience associated 
withactualorpotentialtissuedamageandisessentialtoprotectthe 
bodyfrompotentiallyharmfulstimuli1,2.Chronicpain,however,isnot 
justatemporalcontinuumofacutepainbutoneofthemajorcausesof 
humansuffering,affectingabout20%oftheglobalpopulationwith 
immenseclinicalandeconomicconsequences3,4.Unfortunately,cur- 
rent pain management strategies are largely unsatisfactory, with 
inadequate pain relief and multiple adverse effects5,6. Thus, under- 
standing the mechanisms underlying pain processing remains an 
urgentneedfordevelopingeffectivetreatments.
Accumulatingevidencesupportsthecrucialrolesofmedialpre- 
frontalcortex(mPFC)inregulatingmultipleaspectsofpain,including 
the sensory, affective, and cognitive dimensions7–11. Neuroimaging 
studies in humans have shown functional deactivation and altered 
connectivityofmPFCinpatientswithchronicpain12–14.Consistently, 
preclinicalstudieshavealsorevealedthediminishedactivitiesofmPFC 
inseveralformsofchronicpainandthecriticalrolesofmPFCinpain 
relief15–18. The mPFC is widely connected with other brain regions, 
including the periaqueductal gray (PAG), thalamus, amygdala, and 
basal nuclei19. As part of the descending modulation pathway that 
governsthenoxiousinputsinthespinalcord,themPFC-to-PAGcircuit 
inpainprocessinghasbeenwelldocumented5,16,17.However,itismuch 
lessclearabouttherolesofthemPFCoutputstootherbrainregionsin 
painregulation,aswellashowthemPFCmodulatestheprocessingof 
noxiousinformationinthebrain.
Thethalamus,amajortargetofthemPFCoutput,servesasakey 
hubforintegratingandprocessingnociceptiveinformationascending 
from the spinal cord and modulates both sensory and affective 
dimensionsofpainthroughthelateralandmedialpathways5,20.Ima- 
gingstudieshavelinkedchronicpaintothalamicalterations,including 
reduced volume, activity, and imbalanced transmitters12,14,21. Recent 
findings also highlight the critical functions of the connections 
betweenthethalamusandothercortices,suchasthemotorcortexand 
ACC, in regulating affective pain22,23. Despite the strong anatomical 
connections between mPFC and thalamus and their critical roles in 
pain and other behaviors such as attention and cognition24,25, the 
specificfunctionsofthemPFC-to-thalamuscircuitinpainprocessing 
remainlargelyunexplored.
ThemPFCreceivesinputsfrommultiplebrainregions19.Among 
these,thestrengthenedglutamatergicinputfrombasolateralamyg- 
dala(BLA),whichsynapsesontotheinhibitoryneuronsinmPFCand 
sends feedforward inhibition to the pyramidal neurons, has been 
reportedasakeymechanismunderlyingthedeactivationofmPFCin 
chronicpain17,18.However,therolesofothermPFCinputsinchronic 
painremainlargelyunknown.Notably,themPFCreceivesdensecho- 
linergicinnervationfromthebasalforebrain,apathwayinvolvedin 
variousfunctionssuchaslearning,attention,andarousal26.Although 
theclinicalimportanceofcholinergicsignalinginpainhasbeenwell 
recognized27, the roles and the circuit mechanism of cholinergic 
modulationofmPFCinpainarenotfullyunderstood.Moreover,the 
mPFCishighlyheterogeneous,withdifferentialstructuralandfunc- 
tionaldysregulationofthemPFCinchronicpain8.Thevariablefunc- 
tional responses, either pain relief or exacerbation following mPFC 
activation8,28,29, might resultfrom the cell heterogeneity acrosssub- 
regions and layers of mPFC, which requires higher cell-type and 
projection-specificresolutiontoaccuratelydelineate.
Inthisstudy,weidentifyFoxp2asamarkerofthemPFCneurons 
thatprojecttothethalamusandshowthattheseneuronsaredeacti- 
vatedunderacuteandchronicpain.Usingcell-type-specificand 
circuit-specificmanipulations,wedemonstratethatactivationofthe 
mPFCFoxp2+neuronsalleviatesboththesensoryandaffectivecom- 
ponentsofpainbytargetingdistinctthalamicsubregions.Addition- 
ally, we show that the mPFC Foxp2+ neurons receive cholinergic 
innervationfromthebasalforebrain,particularlythehorizontaldiag- 
onalbandofBroca(HDB),andthatactivationofthecholinergicpro- 
jection is antinociceptive. Notably, we found that activation of the 
α4β2-containing nicotinic acetylcholine receptor (nAChR) in mPFC 
relievespain,whichrequirestheFoxp2+neuronalactivity.Collectively, 
our study reveals a mechanism by which the mPFC regulates pain 
throughprojectiontothethalamus,andhighlightsmPFCcholinergic 
signalingasapotentialtherapeutictargetforchronicpaintreatment.

Results
Foxp2specificallymarksthemPFCoutputstothethalamus
Thecerebralcortexisorganizedintolayerswithdiverseneuronsubtypes
thathavedistinctprojectionpatternsdistributedinalaminarpattern30.In
themPFC,theprojectionstothethalamus,ahubforpainprocessing,
primarilyoriginatefromthelayer(L)6corticothalamic(CT)neurons31,32.
Surprisingly,single-moleculefluorescenceinsituhybridization(smFISH)
revealedthatNtsr1,awidelyusedL6CTneuronmarkerinthecortex22,33,34,
isbroadlyexpressedfromL2toL6inthemPFC(SupplementaryFig.1a),
indicating the different organization of the mPFC compared to other
partsofthecortex.Integrativeanalysisofourprevioussingle-cellRNA
sequencingandspatialtranscriptomicdataindicatedFoxp2andOprk1,
respectively,marktheL6CTandL6intratelencephalic(IT)neurons35,36,
thetwomajorexcitatoryneurontypesinL6(SupplementaryFig.1b).This
is consistent with previous studies suggesting exclusive expression of
Foxp2inCTneuronsofthesensorycortex33,37–40.
To determine whether Foxp2 can serve as a marker for L6 CT
neuronsinthemPFCofadultmice,weinjectedcholeratoxinsubunitB
(CTB) 555, a retrograde tracer, into the medial thalamic nuclei and
performedsmFISHforFoxp2inthemPFC(Fig.1a).Wefoundthatthe
CTB+ cellsare mainlylocated in L6 with over 90% of them express
Foxp2,while around 90% ofthe Foxp2+ neurons are co-labeled with
CTB(Fig.1a),indicatingthattheFoxp2+neuronsaccountforthemajor
outputsofmPFCtothalamus.ToexaminewhetherthemPFCFoxp2+
neuronsprojecttoothermajormPFCtargets,CTBwasalsoinjected
into the PAG (Fig. 1b), contralateral mPFC (Supplementary Fig. 1c),
amygdala (Supplementary Fig. 1d), or nucleus accumbens (NAc)
(SupplementaryFig.1e),respectively.MinimalcolocalizationofCTB
andFoxp2wasobservedintheseconditions(Fig.1bandSupplemen-
taryFig.1c–e).ThesefindingsconfirmthatFoxp2specificallymarksthe
majoroutputsofmPFCtothethalamus.
GiventhattheoutputsofmPFCtothethalamusmainlyoriginate
from the L6 CT neurons, we then asked whether Foxp2 specifically
marks this distinct neuronal subtype. To this end, we performed
smFISHforFoxp2,Slc17a7(amarkerforglutamatergicneurons),and
Gad1(amarkerforinhibitoryneurons),whichrevealedthatover95%
ofFoxp2+cellsexpressSlc17a7(Fig.1c),indicatingthatFoxp2+cellsare
predominantly glutamatergic neurons. To further characterize the
distribution of Foxp2+ cells in the mPFC, smFISH for Foxp2, Etv1 (a
markerforL5),andCtgf(amarkerforL6b)wasperformedwiththe
mPFCsectionsalongtheanterior-posterioraxis(Fig.1dandSupple-
mentaryFig.2).Quantificationofthefluorescencesignalsconfirmed
theexpressionofFoxp2inL6,includingL6b,withabout10%ofFoxp2+
cellsalsoexpressingEtv1(Fig.1dandSupplementaryFig.2a–c).Fur-
therquantificationofthefluorescencesignalsinthemPFCfrompiato
whitematterrevealedthattheFoxp2+cellsaremainlylocatedinL6and
toamuchlessextentindeepL5,especiallyintheanteriorpartofmPFC
(Supplementary Fig. 2d). Since Foxp2 and Oprk1 are indicated to
respectivelymarktheL6CTandITneurons(SupplementaryFig.1b),
we examined their expression in the mPFC and found that they label
twodistinctneuronalpopulationsinL6withminimaloverlap(Fig.1e).
BecausetherearealsoasmallnumberofFoxp2+neuronsinL5,where
residestheL5ITneurons,aswellastheextratelencephalic(ET)neu-
ronslabeledbyPou3f135andnear-projecting(NP)neuronslabeledby
Tshz241,wethenperformedsmFISHandfoundlittleoverlapbetween
Foxp2andPou3f1(Fig.1f)orTshz2(Fig.1g).Collectively,theseresults
indicatethatFoxp2labelsadistinctneuronalsubtypeinL6different
fromtheIT,ETandNPneuronsinthemPFC,consistentwiththetra-
cingresults.
To further confirm that the mPFC Foxp2+ neurons specifically
project to the thalamus, Cre recombinase (Cre)-dependent adeno-
associated virus (AAV) encoding mGFP and synaptophysin-fused
mRuby that labels axon terminals was injected into the mPFC of
Foxp2-Cremice(Fig.1h).Thevirusexpressionwasconfirmedbypost
hochistologicalexamination(SupplementaryFig.1f).Consistentwith
thefactthatFoxp2labelstheL6CTneuronsinthemPFC,wholebrain
mappingrevealedthatthemPFCFoxp2+neuronsprimarilyprojectto
thalamus,withthehighestintensityofmRubysignalsinparataenial
thalamic nucleus (PTN), mediodorsal (MD) and ventromedial (VM)
thalamus (Fig. 1h). Weaker signals were observed in the nucleus
reuniens (Re), paraventricular nucleus of thalamus (PVT) and the
medial striatum (Str), while no signal was detected in other down-
streamtargetsofmPFCsuchasNAc,amygdala,PAG,hippocampus,
hypothalamusandventraltegmentalarea(VTA)(Fig.1handSupple-
mentary Fig. 1g–m). Quantification of the average axon-labeling
mRuby signals indicated that the medial thalamic nuclei, including
PTN, MD, and VM, are the main downstream targets of the mPFC
Foxp2+neurons(Fig.1i).Collectively,theseresultsdemonstratethat
Foxp2specificallylabelsthemPFCoutputstothalamus(Fig.1j).
ThemPFCFoxp2+neuronsaredeactivatedinacuteand
chronicpain
TodeterminewhetherthemPFCFoxp2+neuronsareinvolvedinpain
processing,invivominiscopiccalcium(Ca2+)imagingwasperformed
tomonitortheactivityoftheindividualmPFCFoxp2+neuronsunder
variouspainstates.Tothisend,Cre-dependentAAVencodingtheCa2+
indicator GCaMP7s was injected into the mPFC of Foxp2-Cre mice,
followedbytheimplantationofagradientrefractiveindex(GRIN)lens
abovetheinjectionsitetwoweekslater(Fig.2a).Virusexpressionand
properGRINlensimplantationwereconfirmedbyposthochistological
examination(Fig.2a).Fourweeksafterthevirusinjection,fluorescence
signalsoftheindividualFoxp2+neuronswererecordedthroughthe
GRIN lens using a head-mounted miniaturized microscope, and the
Ca2+signaltracesofindividualneuronswereidentifiedandextracted
(Fig. 2b). All the mice were subjected to pinch, complete Freund’s
adjuvant(CFA)andformalintreatmentswiththeCa2+signalsrecorded
asoutlineinFig.2c.
Toexamine the activitiesoftheFoxp2+ neuronsinresponseto
noxiousmechanicalstimulation,werecordedthebaselineactivityfor
2min,thensubjectedthemicetotailpinchfor1min,andmonitored
anadditional9minforrecovery(Fig.2d).Asshownbytheheatmap
and representative traces, the Ca2+ event frequency of the Foxp2+
neuronsmarkedlydecreasedduringpinchstimulation,followedbya
gradual recovery (Fig. 2e). Quantitative analysis revealed that the
overall Ca2+ event frequency, but not amplitude, across all Foxp2+
neuronsissignificantlydecreasedduringpinchstimulationcompared
to the baseline (Fig. 2f and Supplementary Fig. 3a), indicating the
robustdeactivationofthemPFCFoxp2+neuronalpopulation.Further
analysisofindividualneuronalresponsesshowedthataround80%of
theFoxp2+neuronsexhibitedasignificantdecreaseintheCa2+event
frequency, with 4% of the neurons showing an increase (Fig. 2g).
Together,theseresultsdemonstratethattheoverallFoxp2+neuronal
activityisdecreasedinresponsetonoxiousmechanicalstimulation.
Themicewerealsosubjectedtoformalintreatmentontheindi-
cated day (Fig. 2c, h),which induces biphasic pain responses as pre-
viouslydescribed42.Enhancedcopingbehaviorswereobservedduring
thefirst5min(phase1)andbetween15and30min(phase2)following
theformalininjection(SupplementaryFig.3c).Similartotheresponses
to noxious mechanical stimulation, the Ca2+ event frequency of the
Foxp2+neuronsdecreasedmarkedlyduringbothphase1andphase2,
witharecoverybetweenthetwophases(Fig.2i).Quantitativeanalysis
alsorevealedthattheoverallCa2+eventfrequency,butnotamplitude,
acrossallFoxp2+neuronsissignificantlydecreasedduringphase1and
phase2comparedtothebaseline(Fig.2jandSupplementaryFig.3d,e),
suggestingfunctionaldeactivationofthemPFCFoxp2+neuronalpopu-
lationinresponsetoformalintreatment.Furtheranalysisofindividual
neuronal responses revealed a significant decrease in Ca2+ event fre-
quencyinaround30%ofFoxp2+neuronsandincreaseinaround18%of
theneurons(Fig.2k).Toruleoutpotentialeffectsofpriorpainexperi-
ences,werepeatedtheformalintreatmentusinganewcohortofmice
without preceding stimulation (Supplementary Fig. 3f). We observed
againasignificantdecreaseintheoverallCa2+eventfrequencyofthe
Foxp2+neuronsinbothphase1andphase2(SupplementaryFig.3g–i),
further confirming the deactivation of the mPFC Foxp2+ neurons in
responsetoformalintreatment.Collectively,theseresultsindicatethat
themPFCFoxp2+neuronsaredeactivatedinacutenociception.
Finally,weaskedwhetherthemPFCFoxp2+neuronsareinvolved
inchronicpain.Tothisend,wesubjectedthemicetoCFAtreatmentto
induceinflammatorypain(Fig.2l).TheoverallfrequencyofCa2+events
intherecordedmPFCFoxp2+neuronssignificantlydecreasedinboth
day 1 and day 7 after the CFA injection compared to the baseline
(Fig.2m),indicatingadecreaseintheFoxp2+neuronalactivityinCFA-
inducedinflammatorypain.TobettercomparetheCa2+eventsofthe
individual Foxp2+ neurons among different days, we identified and
analyzedthesameneuronsrecordedindifferentdays(Supplementary
Fig. 3f). We found that a large proportion of the Foxp2+ neurons
exhibited decreased Ca2+ activities on day 1 and day 7 after CFA
injection(Fig.2n,o).QuantitativeanalysisrevealedthedecreasedCa2+
events of the registered Foxp2+ neurons in both day 1 and day 7
(Fig.2p),andthat25.8%and43.5%ofFoxp2+neuronsweresignificantly
deactivatedonday1andday7,respectively(Fig.2q,r).Together,these
resultsdemonstratethatthemPFCFoxp2+neuronsaredeactivatedin
CFA-inducedinflammatorypain.
PersistentinactivationofthemPFCFoxp2+neuronsenhances
nociceptivesensitivity,whileactivationoftheseneuronsalle-
viatesdifferentaspectsofpain
GiventhecriticalrolesofmPFCandthalamusinpainprocessingand
theobserveddeactivationofthemPFCFoxp2+neuronsinbothacute
andCFA-inducedinflammatorypain(Fig.2),weaskedwhetherthese
neurons play a causal role in pain regulation. To this end, Cre-
dependentAAVsexpressingEYFPortetanusneurotoxin(TeNT)were
bilaterallyinjected into the mPFC of Foxp2-Cre mice to persistently
blockthesynapticoutputoftheFoxp2+neurons(Fig.3a).Behavioral
testswereconductedfiveweekslater,withvirusexpressionconfirmed
byposthochistologicalexamination(Fig.3a).Wefirstperformedthe
vonFreytest(VFT)tomeasurethemechanicalsensitivityofthemice.
TheTeNTgroupexhibitedasignificantlydecreasedpawwithdrawal
thresholdcomparedtothecontrolgroup,indicatingthatpersistent
inactivationofthemPFCFoxp2+neuronsinducesmechanicalhyper-
sensitivityofthemice(Fig.3b).Similarly,asignificantdecreaseinpaw
withdrawallatencyinthehotplatetest(HPT)wasalsoobservedinthe
TeNTgroup,indicatingthermalhypersensitivityinthesemice(Fig.3c).
Together, these results indicate that persistent inactivation of the
mPFCFoxp2+neuronsenhancesnociceptivesensitivityinmice.
We next asked whether short-term manipulation of the mPFC
Foxp2+ neurons affects the nociception. To this end, we bilaterally
injectedCre-dependentAAVsencodingmCherryorhM3Dq,anactivat-
ingDREADD(designerreceptorsexclusivelyactivatedbydesignerdrug),
into the mPFC of the Foxp2-Cre mice (Fig. 3d). Virus expression was
confirmedbyposthochistologicalexamination(Fig.3d).Threeweeks
later, behavioral assays were performed 20min after intraperitoneal
(i.p.)injectionofclozapine-N-oxide(CNO).InboththeVFTandHPT,the
hM3Dqgroupshowedasignificantincreaseinpawwithdrawalthreshold
andlatency(Fig.3e),indicatingtheantinociceptiveeffectsinducedby
themPFCFoxp2+neuronactivation.ToexaminetherolesofthemPFC
Foxp2+ neurons in coping behaviors, a formalin test was performed,
whichrevealedasignificantdecreaseinlickingdurationinphase2,but
notphase1,inthehM3Dqgroup(Fig.3f),furthersupportingtheanti-
nociceptiveeffectsofthemPFCFoxp2+neuronactivation.
WenextassessedtherolesofthemPFCFoxp2+neuronsinchronic
painbytreatingthemicewithCFAtoinducepersistentinflammatory
pain.VFTandHPTwereperformed2or3daysafterCFAinjection,
whichrevealedsignificantincreasesinpawwithdrawalthresholdand
latencyinthehM3Dqgroup(Fig.3g),indicatingactivationofthemPFC
Foxp2+neuronsalleviatesthemechanicalandthermalhypersensitivity
ininflammatorypain.Todeterminethecontributionoftheseneurons
totheaffectivecomponentsofpain,wesubjectedthemicetoacon-
ditionedplacepreference(CPP)test.Themicewereconditionedwith
salineinonechamberandCNOinanother(Fig.3h).Afterthreedaysof
training, the hM3Dq group displayed a preference for the chamber
pairedwithCNOtreatment,asindicatedbyincreasedtimespentinthe
chamberandahigherCPPscore(Fig.3h),suggestingthatactivationof
themPFCFoxp2+neuronscouldalleviateaffectiveaspectsofinflam-
matorypain.Collectively,theseresultsindicatethatactivationofthe
mPFCFoxp2+neuronsrelievesbothnociceptivehypersensitivityand
negativeaffectofinflammatorypain.
Toevaluatetheeffectsofshort-terminactivation,webilaterally
injectedCre-dependentAAVsexpressingmCherryorhM4Di,aninhi-
bitoryDREADD,intothemPFCoftheFoxp2-Cremice(Supplementary
Fig. 4a). Since the overall activity of the mPFC Foxp2+ neurons has
alreadybeendecreasedunderpainconditions,asshownabove(Fig.2
and Supplementary Fig. 3), the chemogenetic inactivation primarily
targetedtheactivatedsubsetofthemPFCFoxp2+neuronsinresponse
to noxious stimulation. Surprisingly, no significant effects were
observedinthenociceptivesensitivity,copingbehaviors,ornegative
affectofpainasindicatedbyVFT,HPT,formalintest,andCPP test
(Supplementary Fig. 4b-d), indicating that the activated subset of
Foxp2+ neuronscontributesminimallytopainprocessingorthatits
effectsareoverweighedbythepopulation-widesuppressionofFoxp2+
neurons.Theefficiencyofchemogeneticmanipulationwasconfirmed
as the expression of animmediate-earlygene,c-Fos,wasdecreasedor
increasedafterCNOtreatmentinthehM4DiorhM3Dqgroup(Sup-
plementaryFig.4e).
Collectively,theseresultsindicatethatpersistentinactivationofthe
mPFCFoxp2+neuronsenhancesnociceptivesensitivity,whileactivation
oftheseneuronsexertsrobustantinociceptiveeffects,alleviatingboth
thesensoryandaffectivecomponentsofinflammatorypain.
Projection-specificrolesofthemPFCFoxp2+neuronsinreg-
ulatingacuteandchronicpain
HavingdemonstratedthecrucialrolesofthemPFCFoxp2+neuronsin
regulatingpain,wenextinvestigatedtheunderlyingcircuitmechan-
isms.WehaverevealedthatthePTN,MD,andVMthalamicsubregions
arethemajordownstreamtargetsofthemPFCFoxp2+neurons(Fig.1h,
i and Supplementary Fig. 1f–m). Previous studies have revealed the
rolesofMDandVMinregulatingaffective,butnotsensoryaspectsof
pain7,22,23,while the PTN ismuchlessstudied but implicated inreg-
ulatingpainsensitivityinmice43.TodeterminetherolesofthemPFC
Foxp2+projectionsinregulatingpain,Cre-dependentAAVsencoding
EYFPorchannelrhodopsin-2(ChR2)wereinjectedintothemPFCof
Foxp2-Cre mice, and optical fibers were implanted above the PTN
(Fig. 4a),MD (Fig. 4b), or VM (Fig. 4c), respectively, for projection-
specific activation. Virus expression and fiber implantation were
confirmedbyposthochistologicalexamination(Fig.4a–c).Three
weekslater,VFTandHPTwereconducted.Wefoundthatoptogenetic
activationofthemPFCFoxp2+projectiontoPTNsignificantlyincreased
thepawwithdrawalthresholdinVFTandlatencyinHPTcomparedto
the baseline or the control group (Fig. 4d), indicating reduced
mechanicalandthermalsensitivity.Incontrast,activationofthepro-
jectionstoMDorVMhadnosignificanteffectonnociceptivesensi-
tivity (Fig. 4e, f). These results suggest that mPFC Foxp2+ neurons
reducemechanicalandthermalsensitivitythroughprojectingtoPTN,
but not the MD or VMofthethalamus.
Toexaminetherolesoftheprojectionsincopingbehaviorsunder
tonicpain,wesubjectedthemicetotheformalintestwithoptogenetic
activationofthespecificprojections.Wefoundthatactivationofthe
projections to PTN and VM, but not MD, significantly reduced the
licking duration in phase 2, but not phase 1, of the formalin test
comparedtothecontrolgroup(Fig.4g–i).Theseresultsindicatethat
theprojectionsofthemPFCFoxp2+neuronstothePTNandVMreduce
the coping behaviorsof the mice in responseto tonic pain. Collec-
tively, these results demonstrate that the mPFCFoxp2→PTN circuit
modulatesboththenociceptivesensitivityandcopingbehavior,and
themPFCFoxp2→VMcircuitspecificallymodulatesthecopingbehavior,
whilethemPFCFoxp2→MDcircuitisnotinvolvedineither.
Toassesstherolesoftheprojectionsinchronicpain,wetreated
the mice with CFA toinduce inflammatory painand performed the
pain-relatedbehavioralassayswithoptogeneticstimulation(Fig.5a).
TwoandthreedaysaftertheCFAinjection,VFTandHPTwereper-
formed, respectively, to measure mechanical allodynia and thermal
hyperalgesiaininflammatorypain.WefoundthatactivationofthePTN
projectionsignificantlyincreasedthepawwithdrawalthresholdand
latency(Fig.5b),indicatingreducedmechanicalandthermalhyper-
sensitivityofthemice.Incontrast,nosignificantchangeintheVFTand
HPTwasobservedbyactivatingtheMDorVMprojections(Fig.5c,d).
These results demonstrate that the PTN projection is primarily
responsible for the roles of the mPFC Foxp2+ neurons inalleviating
nociceptivehypersensitivityinCFA-inducedinflammatorypain.
To investigate the roles of the projections in regulating
affective aspects of inflammatory pain, we subjected the mice
to the CPP test, pairing one chamber with optogenetic stimulation
(Fig. 5a). The mice with activation of the PTN or MD projections
exhibited a significant increase inthe time spent in the light-paired
chamberaswellasahigherCPPscore(Fig.5e,f),indicatingthealle-
viated negative affect of pain in these mice. Activation of the
VM projection, however, had no significant effects on the
chamber preference (Fig. 5g). These results indicate that both
themPFCFoxp2→PTNandmPFCFoxp2→MDcircuitsalleviatetheaffective
aspectsofinflammatorypain,whilethemPFCFoxp2→VMcircuitisnot
involved.
Collectively, these results demonstrate that while the
mPFCFoxp2→PTNcircuitisinvolvedinregulatingnociceptivesensitiv-
ity,copingbehaviors,andnegativeaffectofpain,themPFCFoxp2→MD
circuit modulates the negative pain affect, and the mPFCFoxp2→VM
circuitregulatescopingbehaviors,respectively.
ThemPFCFoxp2+neuronsreceiveantinociceptivecholinergic
inputsfromHDB
HavingdemonstratedthefunctionaldeactivationofthemPFCFoxp2+
neuronsinpainandtheirpain-relievingeffectsthroughprojectionsto
distinct thalamic nuclei, we next explored the circuit mechanism
regulating the activity of the mPFC Foxp2+ neurons. Previous studies
haveshownthatmPFCreceivesintensecholinergicinnervationfromthe
basalforebrainandisstronglymodulatedbyacetylcholine(ACh)26,44–46.
Notably, cholinergic modulation of the mPFC is severely impaired in
neuropathicpain,whileactivatingthecholinergicprojectionfromthe
nucleus basalis magnocellularis to the mPFC induces antinociceptive
effects47,48.Theseobservationspromptedustoinvestigatewhetherthe
mPFCFoxp2+neuronsaremodulatedbycholinergicinnervation.
Todetermine whetherthemPFC Foxp2+ neuronsreceivedirect
monosynaptic cholinergic inputs, rabies virus (RV)-based mono-
synapticretrogradetracingwasperformed(Fig.6a).Cre-dependent
AAVhelpersencodingTVA-mCherryandrabiesGproteinwereinjec-
tedintothemPFCofFoxp2-Cremice,followedbyEnvA-ΔGrabiesvirus
expressingEGFPinjectedintothesameregion4weekslater.Oneweek
after the RV injection, the virus expression and starter cells were
confirmedinthemPFC(Fig.6a).Brain-wideanalysisrevealedRV-EGFP-
labeledneuronsinseveralbrainregions,includingthebasalforebrain,
insular cortex, thalamus, ventral hippocampus, and basolateral
amygdala, while fewer labeled neurons were also observed in the
hypothalamus,dorsalraphenucleus,andparabrachialnucleus(Fig.6b
andSupplementaryFig.5a,b).Notably,asubstantialnumberofneu-
ronswerelabeledwithRV-EGFPinthebasalforebrain,particularlythe
HDB,withtheRV-EGFP-labeledneuronsdistributedfromtheanterior
totheposteriorpartofHDB(Fig.6b).Furtherimmunostainingshowed
thatmostRV-EGFP-labeledneuronsinHDBalsoexpresscholineacet-
yltransferase(ChAT),amarkerforcholinergicneurons(Fig.6c),indi-
cating the directmonosynaptic cholinergic inputs from HDB tothe
mPFC Foxp2+ neurons. These results suggest that the mPFC Foxp2+
neurons receive monosynaptic inputs from the HDB cholinergic
neurons.
ToexaminethefunctionsoftheHDBinputstothemPFCinpain,
Cre-dependentAAVsencodingEYFPorChR2wereinjectedintothe
HDBoftheChAT-Cremiceandanopticalfiberwasimplantedabove
themPFCregiontoenablespecificactivationoftheHDBChAT→mPFC
circuit(Fig.6d).Thevirusexpressionaswellasthefiberimplantation
wereconfirmedbyposthochistologicalexamination(Fig.6d).Three
weeksafterthesurgery,VFTandHPTwereperformedandasignificant
increaseintheVFTwasobservedinresponsetooptogeneticactivation
ofthecircuit(Fig.6e),indicatingreducedmechanicalsensitivityofthe
mice.However,nosignificanteffectwasobservedintheHPT(Fig.6e),
indicatingthatthebaselinethermalsensitivityisunaffected.Toassess
theroleofthiscircuitincopingbehaviorsundertonicpain,themice
weresubjectedtoformalintest(Fig.6f).WefoundthattheChR2group
ofmiceexhibitedsignificantlyreducedlickingdurationinphase2,but
notphase1,afterformalintreatment(Fig.6f),indicatingthatactivation
oftheHDBcholinergicinputstomPFCreducesthecopingbehaviorsin
mice.Tofurtherevaluatetherolesofthiscircuitininflammatorypain,
the mice were subjected to CFA treatment. VFT and HPT analyses
indicatedthatoptogeneticactivationoftheHDBcholinergicterminals
in mPFC significantly alleviated both the mechanical and thermal
hypersensitivityininflammatorypain,asindicatedbyincreasedpaw
withdrawalthresholdandlatency(Fig.6g).WenextperformedtheCPP
testtomeasurethenegativeeffectofpain,andsurprisingly,nosig-
nificanteffectwasobserved(Fig.6h),indicatingthatactivationofthe
HDBcholinergicinputstomPFCdoesnotalleviatetheaffectivecom-
ponents of inflammatory pain. Together, these results demonstrate
thatthemPFCFoxp2+neuronsreceivedirectmonosynapticcholiner-
gicinnervationfromtheHDB,whichregulatesthenociceptivesensi-
tivityandcopingbehaviors.
ThemPFCFoxp2+neuronshighlyexpressα4β2nAChR,which
facilitatespainrelief
Giventhecriticalrolesofthecholinergicsignalinginpainprocessing
andtheinnervationofmPFCFoxp2+neuronsbycholinergicinputs,we
next sought to identify the specific acetylcholine receptor (AChR)
responsibleformodulatingthemPFCFoxp2+neurons.Inthecerebral
cortex,therearemainlytwotypesofnicotinicacetylcholinereceptors
(nAChR), the α7 (encoded by Chrna7) and the α4β2 (encoded by
Chrna4andChrnb2)nAChRs,andtwotypesofmuscarinicacetylcho-
linereceptors(mAChR),theM1(encodedbyChrm1)andM2receptors
(encodedbyChrm2)46.Tothisend,weperformedsmFISHanalysesfor
thesereceptorsaswellasFoxp2andOprk1.Theresultsshowedthat
over95%oftheFoxp2+ cellsexpressChrna4whileveryfewexpress
Chrna7 (Fig. 7a). Further smFISH analysis of Chrna4 and Chrnb2
revealed that around 93% of the Foxp2+ cells are positive for both
(SupplementaryFig.6a),whilelessthan10%ofOprk1+cellsexpress
both(Fig.7bandSupplementaryFig.6b),indicatingthepredominant
expressionofα4β2nAChRintheFoxp2+neurons.smFISHanalysisof
mAChRsshowedthatChrm1waswidelyexpressedacrossthemPFC,
withexpressioninnearlyallFoxp2+andOprk1+cellsinL6(Fig.7cand
SupplementaryFig.6c),whileminimalexpressionofChrm2inFoxp2+
cells were observed (Fig. 7d and Supplementary Fig. 6d). Notably,
quantitative analysis revealed the selective expression of the α4β2
nAChRsinFoxp2+cellsinmPFCL6,withnearly80%ofα4β2nAChR-
expressingcellsbeingFoxp2+andonlyaminorityisOprk1+(Fig.7e).
Collectively, these results indicate that while α7 nAChR and M2
expressionsarerelativelylowandM1isbroadlyexpressedinmPFC,
the α4β2 nAChR is highly expressed and enriched in the L6 Foxp2+
neuronsofmPFC(Fig.7f).
GiventhecriticalrolesofthemPFCFoxp2+neuronsinpainpro-
cessingandthestrongeffectsofAChinmodulatingneuronalactivity
inmPFC46,wenextaskedwhethertheα4β2nAChRinmPFCregulates
pain.Tothisend,webilaterallyimplantedaguidedcannulaforintra-
cranialdruginfusionintothemPFCofWTmice,confirmedbyposthoc
histological examination (Fig. 7g). We first infused artificial
cerebrospinal fluid (ACSF) or ABT-594, a specific α4β2 nAChR
angonist27,atdifferentdosages(2.5,5,and25pmol/site)intothemPFC
andperformedVFTtomeasurethemechanicalsensitivityofthemice.
WefoundthatABT-594at5or25pmolpersitesignificantlyincreased
thepawwithdrawalthresholdinVFTcomparedtobothbaselineand
the ACSF control (Fig. 7h and Supplementary Fig. 7a), indicating
reducedmechanicalsensitivitybyactivatingtheα4β2nAChR.smFISH
ofFoxp2andcfosafterACSForABT-594(5pmolpersite)treatment
alsorevealedasignificantincreaseinthecfosexpressioninthemPFC
Foxp2+ neurons (Supplementary Fig. 7b), indicating thatABT-594 at
thisdosageissufficienttoactivatethemPFCFoxp2+neurons.
Sinceboththeantinociceptiveeffectandneuronalactivationare
sufficientlyelicited by the treatment of ABT-594 at the dosage of5
pmol per site, this dosage is used in the subsequent experiments.
Similar antinociceptive effects were observed in the hot plate test
(HPT), with increased paw withdrawal latency following ABT-594
treatment(Fig.7h).Totestwhethertheα4β2nAChRisalsoinvolvedin
regulating chronic pain, we treated the mice with CFA to induce
inflammatory pain. We found that ABT-594 treatment significantly
increased the paw withdrawal threshold in VFT and latency in HPT
(Fig.7i),indicatingthetreatmentalleviatedmechanicalandthermal
hypersensitivityininflammatorypain.However,CPPtestsrevealedno
significanteffectofABT-594treatments(5or25pmolpersite)onthe
affective components of pain (Supplementary Fig. 7c–e), indicating
thatABT-594treatmentisnotsufficienttorelievetheaffectivecom-
ponentsofpain.
The high levels ofα4β2nAChRexpression in themPFCFoxp2+
neurons prompted us to askwhether the antinociceptive effects of
ABT-594dependonactivationofthemPFCFoxp2+neurons.Tothis
end,Cre-dependentAAVsencodingmCherryorhM4Diwerebilaterally
injected into the mPFC of the Foxp2-Cre mice, followed by guided
cannulaimplantationforintracranialdrugdelivery(Fig.7j).Thevirus
expressionandthecannulaimplantationwereconfirmedbyposthoc
histological examination (Fig. 7j). Threeweeksafterthesurgery,VFT
and HPT were performed. In both the mCherryand hM4Di groups,
intracranial ABT-594 delivery significantly increased the paw with-
drawalthresholdinVFTandlatencyinHPT(Fig.7k).Notably,systemic
administration of CNO, which selectively inhibits hM4Di-expressing
mPFC Foxp2+ neurons, abolished these effects in the hM4Di group,
while no significant changes were observed in the mCherry group
(Fig.7k).Theseresultsindicatethattheantinociceptiveeffectsofα4β2
nAChRactivationaremediatedbythemPFCFoxp2+neuronactivity.
Collectively, these results demonstrate thatthe α4β2 nAChR is
highlyexpressedinthemPFCFoxp2+neuronsandactivationofthis
receptor is antinociceptive, which depends on the activationof the
mPFCFoxp2+neurons.

Methods
Animals
All experiments were conducted in accordance with the National
InstitutesofHealthGuideforCareandUseofLaboratoryAnimalsand
approvedbytheInstitutionalAnimalCareandUseCommittee(IACUC)
ofBostonChildren’sHospitalandHarvardMedicalSchool.Thewild-
type C57BL/6 mice (Jax, 000664), B6.Cg-Foxp2tm1.1(cre)Rpa/J mice (Jax,
030541) and B6.129S6-Chattm2(cre)lowl/J mice (Jax, 006410) were pur-
chasedfromJacksonLaboratory.Micewerehousedwithlittermates
(3–5mice/cage)ina12-hlight/darkcycle(lightonfrom7:00to19:00)
withfoodandwateradlibitumunlessotherwisespecified.Ambient
temperature (23–25°C) and humidity (55–62%) were automatically
controlled. For the present study, 2–4-month-old male mice were
used,andallmicewererandomlyassignedtodifferentgroups.Allthe
independentbehavioralassayswereperformedonseparatedaysto
minimize stress and avoid potential confounding effects between
experiments.

Single-moleculefluorescenceinsituhybridization(smFISH)and
immunofluorescence(IF)staining
MicewereeuthanizedbyinhalationofCO andtranscardiallyperfused
2
withPBSand4%paraformaldehyde(PFA)beforethebrainswerecol-
lectedandfixedin4%PFAat4°Covernight,followedbydehydration
in 30% sucrose solution for 3 days. The brains were then frozen in
OptimalCuttingTemperatureembeddingmedia(Sakura,#4583),and
coronalsections(14μmforsmFISHand30μmforIF)werecutusinga
cryostat (Leica, #CM3050S). The smFISH was performed using an
RNAscopeFluorescentMultiplexAssaykit(AdvancedCellDiagnostics,
ACD)followingthemanufacturer’sinstructions.Briefly,thebrainslices
werefixedwithfixedwith4%PFAandwashedwithPBS,thendehy-
dratedwithethanolandtreatedwithhydrogenperoxide.Thesamples
werethentreatedwiththetargetretrievalreagentsandproteasefor
10min,afterwhichthesampleswerehybridizedwiththeprobesat
40°C for 2h. Then the signals were amplified using the reagents
includedinthekit,followedbythelabelingofrelativechannelswith
the TSA fluorophores. The samples were treated with the included
blockingbufferbeforeproceedingtoDAPIstainingandimaging.For
thecombinedsmFISHwithCTBretrogradetracing,thebrainsamples
were prepared from the mice with CTB injected into the relevant
regions. The samples were treated following the manufacturer’s
instructions as described above till the labeling with the TSA fluor-
ophorewithoutbeingtreatedwiththeblockingbuffer.Thesamples
werethendirectlystainedwithDAPI,andtheimageswerecaptured
immediately. Probes (ACD) used were as followed: Foxp2 (Cat. No.
428791;Cat.No.428791-C2);Slc17a7(Cat.No.416631-C3);Gad1(Cat.
No. 400951-C2); Ctgf (Cat. No. 314541); Etv1 (Cat. No. 557891-C3);
Oprk1(Cat.No.316111;Cat.No.316111-C2);Tshz2(Cat.No.431061-C3);
Pou3f1(Cat.No.436421-C2);Chrna4(Cat.No.429871-C3);Chrna7(Cat.
No.465161-C2);Chrnb2(Cat.No.449231-C2);Chrm1(Cat.No.495291-
C2);Chrm2(Cat.No.495311).
For IF staining, the brain slices were washed in PBS twice for
10min,followedbyincubationinblockingbuffer(5%goatserum,5%
BSA,and0.1%TritonX-100inPBS)for40minatroomtemperature.
The samples werethen incubated at4°Cfor 24h with the primary
antibodydilutedinPBScontaining1%BSA.Aftertheincubation,the
sampleswerewashedthreetimeswithwashingbuffer(0.1%Tween-20
in PBS) and incubated with the Alexa Fluor-conjugated secondary
antibodies for 2h at room temperature. The sections were washed
threetimes, mounted, and imaged using a Zeiss LSM800 confocal
microscopeorKeyenceBZ-X810microscope.Antibodiesusedwereas
follows:rabbitanti-cFos(1:1000,SynapticSystems,#226003),rabbit
anti-ChAT (1:1000, Millipore, #ZRB1012-25UL), Donkey anti-rabbit
Alexa Fluor 488 (Thermo Scientific, #A21206), Donkey anti-rabbit
AlexaFluor568(ThermoScientific,#A10042).

Stereotaxicsurgeries
Allstereotaxicsurgerieswereperformedusingasmall-animalstereo-
taxicinstrument(DavidKopfInstruments,model940)undergeneral
anesthesia by isoflurane (0.8lmin−1; isoflurane concentration 1.5%)
in oxygen. A feedback heater was used to maintain the body
temperature of the mice. The eyes of the mice were kept moist
using ophthalmic ointment throughout the surgery. A small
craniotomy above the target brain region was performed using a
dentaldrill.ThevirusesandCTBwereinjectedintothetargetregions
usingaglassmicropipette(10–20μmindiameteratthetip)thatwas
connectedtoananoliterinjector(NanojectIII,DrummondScientific,
#3-200-207).Foreachsite,120nlofviruseswereinjectedataflowrate
of1nls−1unlessotherwisespecified,allowinganadditional5minfor
viral particles to diffuse before the pipette was slowly withdrawn.
After the withdrawal, the wound was sutured, and the mice
wereallowedtorecoverinawarmblanketbeforebeingtransferred
tothehousingcages.Themiceweretreatedwithmeloxicam(Covet-
rus,Cat.No.049756)subcutaneouslydailyfor3daysandmonitored
foranother2days.
Thecoordinatesofviralinjectionandimplantationsitesarebased
onpreviousliteratureandTheMouseBraininStereotaxicCoordinates
(thirdedition)relativetothebregma(anterior-posterior,mediolateral,
dorsoventralaxisinmm):mPFC(1.94,0.45,−2.50),PTN(−0.46,0.30,
−3.70),MD(−1.22,0.50,−3.20),VM(−1.34,0.75,−4.20),PAG(−4.75,
0.55, −2.70), NAc (+1.20, 0.60, 4.50), BLA (−1.34, 2.90, −4.50), HDB
(−0.10,1.45,−5.50).

Chemogeneticandoptogeneticmanipulations
ForchemogeneticmanipulationsofthemPFCFoxp2+neurons,AAV5-
hSyn-DIO-mCherry (Addgene, Cat# 50459), AAV5-hSyn-DIO-
hM4D(Gi)-mCherry (Addgene, Cat# 44362), or AAV5-hSyn-DIO-
hM3D(Gq)-mCherry (Addgene, Cat# 44361) was bilaterally injected
into the mPFC of Foxp2-Cre mice. The behavioral assays were per-
formed3weekslater,andthemicewereintraperitoneallyinjectedwith
CNO(Cayman,Cat#16882)at1mg/kg(foractivation)or5mg/kg(for
inactivation)insaline20minbeforeeachbehavioraltestaspreviously
described66,67.ForpersistentinactivationofthemPFCFoxp2+neurons,
rAAV2/9-hSyn-DIO-EYFP (BrainVTA, Cat# PT4250) or rAAV2/9-hSyn-
DIO-TeNT-EYFP(BrainVTA,Cat#PT26697)wasbilaterallyinjectedinto
themPFCofFoxp2-Cremice,andbehavioralassayswereperformed
5weekslater.
ForoptogeneticactivationoftheterminalsofmPFCFoxp2+neu-
rons, rAAV2/9-EF1a-DIO-EYFP (BrainVTA, Cat# PT0012) or rAAV2/9-
EF1a-DIO-ChR2-EYFP(BrainVTA,Cat#PT0001)wasbilaterallyinjected
intothemPFCofFoxp2-Cremice,followedbythebilateralimplanta-
tionofopticalcannulas(diameter:200μm;N.A.,0.37;length,4.0mm;
InperInc.,China)100μmabovethePTN/MData20-degreeangleor
VMata10-degreeangle,andsecuredwithdentalcement(Parkell,no.
S380).ForoptogeneticactivationofthecholinergicinputstomPFC,
thesameviruseswereinjectedintotheHDBregionofChAT-Cremice,
and the optical cannulas were implanted above the mPFC. For all
optogenetic manipulation, the mice were connected to the optical
fiberforacclimationfor3daysbeforethebehavioraltestsandthelight
pulses(473nm,1.5mWatthetipofthefiber,20Hz,5-mspulse)at10-s
ON/OFFcycleweregeneratedbya473-nmlaser(OEMlaser/OptoEn-
gine)andusedthroughoutthebehavioraltestscontrolledbyawave-
formgenerator(Keysight).

Neuronaltracing
ForCTBretrogradetracing,threeinjectionsofCTB-555(200nleach)
(Invitrogen,Cat#C34776)targetingthemedialthalamicnucleiwere
performed to efficiently label the mPFC neurons projecting to the
thalamus.Forotherregions,includingmPFC,NAc,oramygdala,120nl
ofCTB-555wasunilaterallyinjectedinindividualmice,respectively.
Thebraintissueswerecollected1weeklaterforsmFISH.Forspecific
axon labeling of the mPFC Foxp2+ neurons, pAAV1-hSyn-DIO-mGFP-
Synaptophysin-mRuby(Addgene,Cat#71760)wasunilaterallyinjec-
tedintothemPFCofFoxp2-Cremice.
Formonosynapticretrograderabiestracing,a150nlmixtureof
AAVhelpers(rAAV2/9-EF1a-DIO-mCherry-TVAandrAAV2/9-EF1a-DIO-
RVG)(BrainVTA,Cat#PT0023andPT0207)wasunilaterallyinjected
intothemPFCoftheFoxp2-Cremice.Fourweekslater,thesamemice
were injected with 250nl of rabies virus (RV-ENVA-ΔG-EGFP)
(BrainVTA,Cat#R01001)tothesamelocation.Thebraintissueswere
collected7dayslaterforimagingandexamination.

Intracranialdrugdelivery
For intracranial drug delivery into the mPFC, the double cannula
(Guidecannula:M3.5,C.C1.0mm,C=2.5mm;Injector:G1=0.5mm;
Dummycannula:G2=0,mateswithM3.5;RWDLifeScience)withdust
caps targeting both sides wasdirectly implanted 0.5mmabove the
mPFC and was secured with dental cement. Two weeks later, an
injector(G2=0,mateswithM3.5;RWDLifeScience)filledwithmineral
oilandABT-594(CaymanChemical,Cat.No.22822)wasconnectedtoa
pump(RWDLifeScience)toslowlyinfusethedrugintobothsidesof
mPFC(200nl/site,5min)and5minwereallowedfordrugdiffusion
before the injector was slowly removed. The behavioral tests were
performed another 5min later. For smFISH of cfos, the mice were
sacrificed30minafterthedruginjection.Forintracranialdrugdelivery
combinedwithchemogeneticinhibition,AAV5-hSyn-DIO-mCherryor
AAV5-hSyn-DIO-hM4D(Gi)-mCherry was bilaterally injected into the
mPFC, followed by the implantation of the double cannula. Three
weeks later, the behavioral tests were performed 20min after CNO
injection(i.p.)and5minafterABT-594infusion(intracranially).

Invivominiscopiccalciumimaginganddataprocessing
ToprobetheactivityofthemPFCFoxp2+neuronsinresponsetopain,
in vivo miniscopic calcium imaging was performed as previously
described67,68. Firstly, AAV1-hSyn-DIO-jGCaMP7s (Addgene, Cat#
104491)wasunilaterallyinjectedintothemPFCoftheFoxp2-Cremice.
Twoweekslater,themicewereanesthetizedandfixedonastereotaxic
frame.Theskullabovethetargetregionwascarefullyremovedusinga
dentaldrillingandthebraintissueabovethetargetregionwasaspi-
ratedusinga27-gaugeneedlewithablunttip.ThenanintegratedGRIN
lens(diameter1.0mm,length4.0mm;Inscopix)connectedtoadata
acquisitionsystemformonitoringthecalciumsignalwasslowlylow-
ered (~100μm/min) down to 300μm above the target region. The
GRINlenswasfurtherinsertedtoobtainanoptimalfieldofviewto
image calcium signals, and then the GRIN lens was secured to the
mice’s skull with dental cement attached to a baseplate cover for
protection.Salinewascontinuouslyaddedtoavoidbleedingordrying
of the tissue during the surgery. After the surgery, the mice were
individually housed for 2 weeks before being used in imaging
experiments.
Priortocalciumimagingrecording,themicewereconnectedto
themicroscopethroughthehead-fixedbaseplateandacclimatedto
thehandlingoftheexperimenterandthetestingchamberfor30min
eachday for 3 days.On the day of data acquisition, the mice were
connectedtothemicroscopeandallowedtomovefreelyinthetesting
chamber. After 10min of acclimation, the calcium images were
obtainedfor20–30minusingthenVokesystem(Inscopix)at20Hz
with0.6–1.2mWofLEDpowerand1.5ofgain.Forindividualmice,the
imagingparameterswerekeptconsistentacrossdays.
For tail pinch stimulation, after acclimatation, the mice were
placedinthetestingchamberwithoutstimulationfor2minandthen
analligatorclipwasappliedtothetailfor1min,afterwhichtheclipwas
removed, and the mice were allowed to recover for 9min, and the
calciumsignalswererecordedduringtheentireprocess.Forformalin
stimulation,afteracclimatationthemicewererecordedinthetesting
chamberwithoutstimulationfor5min,andthen20μlofformalinwas
injected into the plantar of the mice, after which the mice were
recordedforanother30mininthechamber.Inonecohort(Fig.2),the
miceweresubjectedtopinchstimulation,CFAtreatment,and,4weeks
later,formalintreatment.Inanothercohort(SupplementaryFig.3f–i),
themiceweresubjectedtoformalintreatmentonly.
Todetectthespontaneousactivityoftheneurons,themicewere
acclimated to the microscope and testing environment with fixed
brightness,temperature,andnoiselevelonehourbeforetherecord-
ings. To avoid the potential effects of the light-dark cycle on the
neuronalactivity,alltherecordingswereperformedintheafternoon
and at the fixed time points for each individual animal. After the
acclimation, the calcium activities were recorded for 15min for the
baseline. Then the miceweresubjected to CFAinjection under iso-
fluraneanesthesia.Oneorsevendaysaftertheinjection,thecalcium
activitieswererecordedagainatthesametimepointafteracclimation
withoutanyadditionalstimulation.
Fordataprocessing,thecalciumtraceswereextractedandana-
lyzed using the Inscopix Data Processing Software (version 1.9,
Inscopix). The raw images were cropped, temporally downsampled
(2×),spatiallydownsampled(2×),andfurtherspatiallyfilteredaswell
asmotion-correctedusingthedefaultsettings.Thesignalsofputative
individualcellswereidentifiedusinganextendedconstrainednon-
negative matrix factorization (CNMF-E) algorithm and werenormal-
izedtoΔFovernoise(settings:celldiameter:8pixels;minimumpixel
correlation: 0.85; minimum peak-to-noise ratio: 15). The identified
individualcellswerethenmanuallycheckedtobeincludedbasedon
their anatomy(size,shape,location), signal-to-noise ratio aswellas
overlap in spatial location and signal with other cells. The calcium
signaltraceswerefurtherdeconvolutedusingOASIS(onlineactiveset
method to infer spikes) methods to detect the potential calcium
transientswiththedefaultnoiserangesandspikesignal-to-noiseratio
of8.0.Acrossthedifferentdaysofrecordingforchronicpain,wefirst
analyzed the calcium images to identify individual neurons in each
recordingandcomparedthespontaneouscalciumactivitiesofallthe
identifiedneuronsamongdifferentdays.Thentheidentifiedindividual
neuronswerelongitudinallyregisteredwithaminimumcorrelationof
0.9,andthematchedneuronswerefurthermanuallycheckedtobethe
sameneuronsacrossdifferentdaysbasedonthelocationsandother
visiblelandmarks(suchasbloodvessels).
Toidentifytheneuronswithsignificantchangeinthefiringratein
responsetotailpinchstimulation,thecalciumtraceswereextracted
anddeconvolutedusingOASIStoidentifythecalciumtransients.We
pooled together the identified transients of 2min before and 1min
duringthetailpinchstimulation,andusedapermutationmethodto
create10,000shuffleddistributionsofthetransients.Thentheshuf-
fled transient change was calculated by subtracting the shuffled
0–2minfromthatofthe3rdmin.Theneuronswithactualtransient
changerankedhigherthan99.5thpercentileoftheshuffledtransient
changewereclassifiedasactivity-increasedneurons,andthoseranked
lowerthan0.5thpercentilewereclassifiedasactivity-decreasedneu-
rons.Fortheformalintest,thecalciumsignalsof−5minto0minand
those of 0min to 5min were compared. For CFA-induced chronic
inflammatorypain,the calciumsignalsofthebaseline,CFAD1,and
CFAD7werecomparedusingthesamemethod.

Behavioralassays
von Frey test. The mechanical sensory threshold of the mice was
measuredwithaseriesofvonFreyfilaments(rangingfrom0.04to
2.0g,Stoelting)usingtheUp-Downmethodaspreviouslydescribed69.
Briefly,themicewereplacedinaplasticchamberwithametalmesh
floorandallowedtomovefreely.After20minofacclimationofthe
mice,thevonFreyfilamentswereappliedtothemid-plantarsurfaceof
thehindpawthroughthemeshfloorandkeptfor2s,oruntiltherewas
apositiveresponse(pawwithdrawal,flinching,licking,orshaking).To
avoidthepotentialfalsepositiveresponsescausedbymovement,the
measurementwasconductedagainifapositiveresponsewasfollowed
by other voluntary behaviors, such as locomotion, exploratory, or
groomingbehaviors.

Hotplatetest.Thehotplatetestwasusedtomeasurethermalnoci-
ception ofthe mice. Afterbeingacclimated inthetesting roomfor
20min,themicewereplacedonahotplate(UgoBasile)setat52°C,
andthelatenciestoflinchingorlickingofhindpawsweremeasured.All
animalsweretestedfor3timeswithaminimumintervalof30min.To
avoidtissueinjury,acutofflatencywassetat30sforthetest.

Formalintest.Formalintestwasperformedaspreviouslydescribed42.
Briefly,20μlof2%formalin(Sigma-Aldrich,HT5011-1CS)wasinjected
subcutaneouslyintotheplantarsurfaceofonehindpawofthemice
witha30-gaugesyringeasquicklyaspossible.Thelickingbehaviors
weremeasuredfor45minstartingfromtheinjectionofformalin,and
thelickingdurationwascalculatedin5-minbins.Thelickingduration
ofthemiceduringphase1(definedas0–5minafterformalininjection)
and phase 2 (defined as 15–45min after formalin injection) was
analyzed.

Complete Freund’s adjuvant (CFA) injection. For CFA-induced
inflammatory pain, 15μl of CFA (Sigma, F5881-10ML) was sub-
cutaneouslyinjectedintotheplantarsurfaceoftherighthindpawof
themiceunderbriefisofluraneanesthesia.

Conditioned place preference. The conditioned place preference
testwasusedtomeasurethenegativeaffectofpainandtheeffectsof
painreliefaspreviouslydescribed67.Thetestingapparatusconsistsof
an infrared photobeamdetectorfor tracking the mice's trajectories
andtwocompartments,oneofwhichiswithrodstylefloorandthe
other has a grid-style floor (Med Associates). For chemogenetic
manipulations, individual mice were placed in the chamber and
allowedtofreelyexploretheentireapparatusfor30min(pretest)on
day1.Ondays2–4,miceinjectedwithsalinewereplacedinthecom-
partmentwithagridfloor(unconditionedcompartment)for30minin
themorning,whilemiceinjectedwithCNOwereplacedintheopposite
compartmentwitharodfloor(conditionedcompartment)ofthesame
apparatusfor30minintheafternoon(withaminimumintervalof6h
between the two conditioning). On day 5,like day1,themicewere
placedinthechamberandallowedtofreelyexploretheentireappa-
ratusfor30min(posttest).Foroptogeneticactivation,thepretestand
posttestonthefirstandlastdayweresimilarlyperformed.Ondays
2–4,themicewereconnectedwithanopticalfiberwithoutlightsti-
mulationandplacedinthecompartmentwithagridfloorfor30minin
themorning,andintheafternoon,themicewereconnectedwiththe
opticalfiberwithlightstimulationasdescribedabove,andplacedin
thecompartmentwitharodfloorfor30min.ForABT-594treatment,
thepretestandposttestonthefirstandlastdayweresimilarlyper-
formed.Ondays2–4,themicewereintracraniallyinjectedwithACSF
and placed in the compartment with a grid floor for 30min in the
morning,andintheafternoon,themicewereinjectedwithABT-594
andplacedinanothercompartmentfor30min.TocalculatetheCPP
score, the time that mice spent in the conditioned compartment
during pretest was subtracted from the time they spent during
posttest.

Statistics
Themicewererandomlyassignedtothegroups,andtheinvestigators
wereblindtotheallocationofthegroupsortheoutcomeassessment.
Nostatisticalmethodwasusedtopredeterminethesamplesize,but
oursamplesizesaresimilartothosereportedinpreviouspublications.
Micethat,afterhistologicalinspection,hadthelocationoftheviral
injection(reporterprotein),cannulaimplantation,ortheopticfiber(s)
outsidetheareaofinterestwereexcluded.Thedataarepresentedas
means±SEM. Statistical analyses were performed using GraphPad
Prism (version 8.0). Student’s t-test and one-way or two-way RM
ANOVA test with Tukey’s or Sidak’s post hoc multiple comparisons
were applied to determine statistical differences. Statistical sig-
nificancewassetat*P<0.05,**P<0.01,***P<0.001.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Prefrontal cortex (PFC) is a well-known brain region to govern pain states and the layer 5 of mPFC has been well established to encode pain signaling. This manuscript identifies deactivation of specific Foxp2 neurons in the layer 6 of mPFC to regulate sensory pain and affective pain by projection to different subregions of thalamus. Furthermore, they traced cholinergic neurons from HDB of basal forebrain as up-stream inputs to mPFC and activation of α4β2 nAChR in the mPFC is analgesic. Therefore, they revealed that the HDBChAT→mPFCFoxp2→thalamic nuclei circuit is crucial for pain modulation.\nOverall, the manuscript provides some insights and extends the understanding of mPFC in pain regulation in the field. The manuscript is well written, however, some of the experiments are not rigorous designed which weakens the conclusion. I have some major and minor concerns to be addressed before considering publishing.\nMajor concerns:\n1, For Fig 1a, the authors claim that around 63.2% of Foxp2+ neurons co-stained with CTB555, however, it seems non-CTB Foxp2+ neurons are more than the CTB-labelled neurons in the image. The authors may show more representative images to support this.\n2, Similarly in Fig 1c, there are a lot of Foxp2+ neurons not labelling with Slc17a7 and it is hard to believe that the quantification is more than 90% as the authors claimed.\n3, From Fig 2c, it seems the pinch, formalin test and CFA were performed in the same batch of mouse? Do the authors perform CFA first and then formalin model? How many mice were used per group? These should be clearly stated in the main text for figure legends.\n4, In figure 2, the deactivation of Foxp2+ neurons is robust in the pinch stimulation, however, this deactivation is a bit low in formalin model (30%), with 17.66% increased and 51.4% no change, not like what the authors claimed that “a large subset was decreased” in line 193-197. So, it is hard to say what the real effect of these neurons are. Similarly, the deactivation is a bit low (25.8% and 43.5%) in the CFA model. The authors should use other methods like c-Fos staining or e-phys recording to confirm these results.\nIn addition, what causes the huge difference of deactivation of Foxp2+ neurons between the pinch and inflammatory pain models? The authors seem to ignore the increased activation and no changes neurons which take more amount than the deactivated one. What does the increased activation of neurons do?\n5, In fig 3g, why the activation of Foxp2+ neurons were tested on day 2 and day 3 after CFA injection rather than day 1 and day 7 with deactivation of Foxp2+ neurons shown in Fig 2p-r. Do the authors confirm that these neurons were really activated by CNO in the CFA model? The quality of image is poor/over-exposed in Fig S4e and it is hard to see the signals. The author should quantify that virus-expressed c-Fos to confirm these neurons are activated rather than c-Fos only. Also, it is better to quantify the number normalized to sample size rather than the number only.\n6, Fig 4a/b: Do the authors implant fibers with angle? There is no description in methods.\n7, There are much more monosynaptic input neurons from IC, VM and BLA to the PLPFC than HDB in Fig. 6b and Fig. S5a, b. Do the authors check any of them to see whether these circuits impact nociceptive responses? Does HDB-PLPFC circuit influence affective pain? Basal ganglia and striatum also express cholinergic neurons. Do the authors checked these brain regions having inputs to mPFC?\n8, In Fig6f, why the mechanical and thermal pain was tested on different day?\n9, In Fig7j, the track is far away from the virus expression, the drug effect is questionable. The authors should provide evidence to show that ABT-594 can activate Foxp2+ neurons in the mPFC by e-phys recording or at least c-Fos staining.\nMinor:\n1, For all behavioral responses with mechanical and thermal stimuli, like Fig 3e, 3g, Fig 4d/e/f , Fig 5b/c/d and etc, the Y axis should be fully described with Paw withdrawal threshold and Paw withdrawal latency.\n2, No scale bar in Fig S3f.\n3, Line 264: c-Fos missed a dash\n4, The two phases of formalin model are 0-5min and 15-45min, the dash square in Fig3f, Fig 4g/h/i is not accurate.\n5, In the manuscript, the authors use PLmPFC and mPFC in different place. Please keep consistent.\n6, Fig 7k, the Y axis should be Paw withdrawal latency for HPT.", "reviewer_2": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_3": "In the article titled “A molecularly defined basalo-prefrontal-thalamic circuit regulates sensory and affective dimensions of pain”, Xie et al. report that a population of Foxp2-expressing neurons in mPFC layer 6 corticothalamic neurons. These neurons exhibit suppressed activity during pain states, and their specific activation significantly alleviates pain symptoms, including hyperalgesia and affective pain components. Furthermore, using viral tracing and immunostaining, the authors demonstrated that Foxp2 neurons project to distinct downstream thalamic nuclei to independently regulate sensory and affective pain dimensions. Finally, they revealed that these Foxp2 neurons receive inputs from HDBChAT neurons; activation via α4β2 nAChRs induces analgesia, thereby delineating an HDBChAT→mPFCFoxp2→thalamus neural circuit governing distinct pain components. Overall, this study demonstrates strong innovation and completeness, supported by robust experimental evidence and rigorous conclusions. However, there are several concerns with the findings that need to be addressed before consideration for publication.\nMajor Points:\n1. Following CFA injection to induce inflammatory pain in figure 2C, the authors did not disclose whether formalin testing was performed on the same cohort of mice. Prior pain experiences—even after full recovery—could confound conclusions from subsequent formalin imaging recordings (such as pain memory effects).\n2. The rationale for inhibiting Foxp2 neurons specifically in formalin-induced pain model mice is unclear (Figure S4). Since Foxp2 neuronal activity is already suppressed in pain states, chemogenetic inhibition in this model is functionally uninformative. This experiment should instead be conducted in naive mice.\n3. The authors found Foxp2 primarily co-localized with L6 glutamatergic neurons. They should discuss how mPFC L6 glutamatergic neurons projecting to different thalamic nuclei (PTN, MD, VM) regulate distinct pain dimensions, citing existing literature. This comparison is essential to establish the specific regulatory role of Foxp2+ neurons versus broader L6 glutamatergic populations in pain processing.\n4. This paper reported three key findings: 1) Deactivated activity of mPFC L6 Foxp2 neurons in pain states; 2) Activation of Foxp2→thalamus projections alleviates pain phenotypes; 3) HDBChAT→mPFCFoxp2 inputs induce analgesia via α4β2 nAChRs. Critical unresolved questions weaken the mechanistic narrative: How does initial pain signaling suppress mPFC Foxp2 activity? Under what conditions does the HDBChAT→mPFCFoxp2 circuit exert analgesia? How is HDBChAT neuronal activity altered during pain? For example, does this circuit function as an endogenous analgesic system during CFA-induce pain recovery? If inhibited, would pain persistence be prolonged?\nMinor Points:\n1. CFA-induced pain (7 days) is inappropriately labeled “chronic”, even Lines 302–305: two and three days after CFA injection... in chronic inflammatory pain. It is clearly inappropriate to refer to such a short period of time as chronic pain, please revise these descriptions.\n2. Provide complete details in all figure legends, now sample size is not clarification. For example, Fig 2D: “n=280” clarify if this represents cells and specify the number of mice.\n3. Explicitly label mice as CFA model mice in the figure/legend, such as Figure 3H.\n4. For formalin licking duration (Phase I/II), use two-way ANOVA to demonstrate statistical significance.\n5. Lines 371–375, Ambiguous description of nAChR subtype-encoding genes. Clarify.", "reviewer_4": "This study revealed new insight into the role of mPFC in regulating different dimensions of pain. They found that Foxp2 expression marks a group of layer 6 mPFC neurons that project to several medial thalamic nuclei and are deactivated by acute noxious stimuli and by chronic inflammatory conditions. Loss- and gain-of functional studies showed that inactivation of these neurons are crucial for driving nocifensive reactions, coping responses and affective pain. Interestingly, these distinct functions are apparently mediated via Foxp2+ neurons that project to distinct thalamic nuclei. Finally, they show that cholinergic inputs to Foxp2+ neurons, via activation of the a4b2 nicotinic acetylcholine recepotors, can modulate nociception, but less on affective pain. Most conclusions are well supported by the data and the finding is interesting, with potential therapeutic impact.\nI have only a few comments:\nTO suggest that cholinergic signaling at mPFC has therapeutic potential, they need to assess the impact on affective pain in response to activation of cholinergic terminals from HDB brain regions, which had not been done in Figure 7. The CPP data described in Extended data Fig 7 did not reveal a robust impact on affective pain. Do the authors need to try different doses of the drug?"}, "author_response": "Responses to Reviewers’ Comments\nWe thank the reviewers for their critiques and helpful suggestions. To address their questions, we\nhave performed a series of experiments and tried our best to improve the clarity of the manuscript.\nOur point-by-point response to the reviewers’ questions are below:\nReviewer #1: ... [Full text of author response provided in the prompt]"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The all authors addressed most of my concerns and the paper is significantly improved. I have no more comments.", "reviewer_3": "The authors have addressed my concerns and the manuscript has been substantially improved. No more questions.", "reviewer_4": "Tha authors have carefuly addressed the issues I raised. While the new CPP studies suggest the involvement in coping\nlicking responses, but not affective pain and therefore reduced the potential clinical impact, the overall finding is still\ninteresting."}, "author_response": ""}]

Decision letter