Role of purines in brain development, from neuronal proliferation to synaptic refinement

Neu opha macology 237 (2023) 109640 A ailable online 20 June 2023 0028-3908/© 2023 The Au ho s. Published by Else ie L d. This is an open access a icle unde he CC BY license (h p://c ea i ecommons.o g/licenses/by/4.0/). Role o pu ines in b ain de elopmen , om neu onal p oli e a ion o synap ic e inemen Solen Rimbe a , Jo˜ ao B. Mo ei a a , b , c , Sa a Xapelli b , c , Sabine L´ e i a , * a INSERM UMR-S 1270, So bonne Uni e si ´ e, Ins i u du Fe ` a Moulin, 75005, Pa is, F ance b Ins i u o de Fa macologia e Neu ociˆ encias, Faculdade de Medicina, Uni e sidade de Lisboa, Lisboa, Po ugal c Ins i u o de Medicina Molecula - Jo˜ ao Lobo An unes (iMM - JLA), Faculdade de Medicina, Uni e sidade de Lisboa, Lisboa, Po ugal ARTICLE INFO Keywo ds: Pu ine gic signaling P1 ecep o s P2 ecep o s ATP Adenosine De elopmen Neu ogenesis Synap ogenesis ABSTRACT The pu ine gic sys em includes P1 and P2 ecep o s, which a e ac i a ed by ATP and i s me aboli es. They a e exp essed in adul neu onal and glial cells and a e c ucial in b ain unc ion, including neu omodula ion and neu onal signaling. As P1 and P2 ecep o s a e exp essed h oughou emb yogenesis and de elopmen , pu i- ne gic signaling also has an impo an ole in he de elopmen o he pe iphe al and cen al ne ous sys em. In his e iew, we p esen he exp ession pa e n and ac i i y o pu ine gic ecep o s and o hei signaling pa h- ways du ing emb yonic and pos na al de elopmen o he ne ous sys em. In pa icula , we e iew he in ol emen o he pu ine gic signaling in all he c ucial s eps o b ain de elopmen i.e. in neu al s em cell p oli e a ion, neu onal di e en ia ion and mig a ion as well as in as ogliogenesis and oligodend ogenesis. Then, we e iew da a showing a c ucial ole o he ATP and adenosine signaling pa hways in he o ma ion o he pe iphe al neu omuscula junc ion and o cen al GABAe gic and glu ama e gic synapses. Finally, we examine he consequences o de egula ion o he pu ine gic sys em du ing de elopmen and discuss he he apeu ic po en ial o a ge ing i a adul s age in diseases wi h eac i a ion o he ATP and adenosine pa hway. 1. In oduc ion The de elopmen o an o ganism is a complex p ocess o cellula and molecula in e ac ions, highly egula ed and ime-p ecise. The o ma- ion o he cen al ne ous sys em (CNS) includes p oli e a ion o neu al s em cells (NSCs) ha di e en ia e in o neu onal and as oglial lineages, ha a e equi ed o o ganize and in eg a e in an in ica e s uc u e (G¨ o z and Hu ne , 2005). Mo eo e , he ma u a ion o newbo n neu ons in- cludes he o ma ion o dend i ic spines and synapses in a p ocess named synap ogenesis (Südho , 2018). Due o i s immense in icacy, he de elopmen al p ocess occu s unde igh egula ion by a ple ho a o egula o y mechanisms, including by he pu ine gic sys em. He e we will de ail he con ibu ion o pu ines, hei ecep o s and signaling pa hways in b ain de elopmen . We will i s ly desc ibe he pu ine gic ecep o s exp essed, some ansien ly, du ing de elopmen . Then, we will discuss he con ibu ion o he pu ine gic sys em in all s eps o neu onal de elopmen (i.e. neu ogenesis, neu onal mig a ion, axon and dend i e a bo iza ion, synap ogenesis), as well as in as ogliogenesis and oligodend ogenesis. 2. Exp ession o pu ines and pu ine ecep o s du ing de elopmen 2.1. Pu ines All b ain cells p oduce and elease pu ines, such as adenosine iphospha e (ATP) and i s me aboli es adenosine diphospha e (ADP), adenosine monophospha e (AMP) o adenosine, which ha e essen ial physiological unc ions. ATP is known o cons i u e he cellula sou ce o ene gy, bu i also has a ole in cellula in e ac ion and communica ion. In he CNS, ATP can be s o ed alone o wi h neu o ansmi e s in esicles and can be eleased by glial cells h ough exocy osis (Zhang e al., 2003) o by neu ons as a co- ansmi e oge he wi h glu ama e o γ-bu y ic acid (GABA) a exci a o y glu ama e gic and GABAe gic syn- apses. Pu ines can be exocy osed in esponse o elec ical s imula ion, glu ama e ecep o agonis o in case o inju y (Pank a o e al., 2006, 2007; Wall e al., 2013). In neu ons, he amoun o ATP in synap ic esicles is compa able o GABA and glu ama e sugges ing an impo an ole o his nucleo ide in synap ic unc ion (Zisapel and Zu gil, 1979). As ocy es also la gely con ibu e o ex acellula ATP, ia o he * Co esponding au ho . E-mail add ess: [email p o ec ed] (S. L´ e i). Con en s lis s a ailable a ScienceDi ec Neu opha macology jou nal homepage: www.else ie .com/loca e/neu opha m h ps://doi.o g/10.1016/j.neu opha m.2023.109640 Recei ed 29 Ap il 2023; Recei ed in e ised o m 15 June 2023; Accep ed 19 June 2023 Neu opha macology 237 (2023) 109640 2 mechanisms han exocy osis. S ill con o e sial, se e al s udies p opose ATP elease by as ocy ic junc ions o med by connexins (Kang e al., 2008) o pannexins (Iglesias e al., 2009) bu also by anion channels such as olume- egula ed anion channels (VRAC) (Blum e al., 2010) and Maxi anion channels (Zhao e al., 2017). I has been p oposed ha adenosine may be exocy osed in an ac ion po en ial- and calcium-dependen manne by hippocampal neu ons (Wall e al., 2013). Howe e , o he da a showed ha educing ex acellula calcium le els in hippocampal slices inc eases adenosine elease om equilib a i e nucleoside anspo e s (ENTs) (Diez e al., 2017; Baldwin e al., 2004). Ex acellula and in acellula ATP is apidly hyd olyzed by en- zymes localized on glial cells and neu onal memb anes (Bjelobaba e al., 2007). In a hippocampal slices, ex acellula ATP deg ada ion oc- cu s wi hin milliseconds (Dunwiddie e al., 1997) h ough a channeling p ocess allowing a local inc ease in adenosine (Cunha e al., 1998). ATP is me abolized in o AMP by CD39 ec onucleo idase and hen in o adenosine by CD73 ec onucleo idase. The ec onucleo idases CD39 and CD73 a e de ec ed a synapses du ing he pe iod o synap ogenesis, allowing local pu ine gic ecep o ac i a ion a he synapse (G ko i´ c e al., 2019). Mo eo e , a ansien inc ease in e oked ac i i y-dependen elease o ATP and adenosine has been shown du ing he pe iod o synap ogenesis (i.e. a pos na al day 7, P7) as compa ed o adul s age (P60) in he oden hippocampus (Gomez-Cas o e al., 2021), sugges ing a ole o his signaling pa hway in b ain de elopmen . Once in he ex acellula space, pu ines ac i a e pu ine gic ecep o s composed o 2 sub amilies: P1 ecep o s (P1R) and P2 ecep o s (P2R) (see below). 2.2. P1 ecep o s P1 ecep o s a e G p o ein coupled ecep o s (GPCRs) ha p e e - en ially inc ease o dec ease cAMP p oduc ion ia he ac i a ion o in- hibi ion o adenylyl cyclases (ACs) depending on he na u e o he G p o ein o which hey a e coupled (F edholm e al., 1994). Since aden- osine is he main agonis o P1 ecep o s, he la e a e known as adenosine ecep o s. These a e di ided in o ou sub ypes, he A 1 (A 1 R), A 2A (A 2A R), A 2B (A 2B R) and A 3 (A 3 R) ecep o s, which ha e a high de- g ee o s uc u al homology. A 1 R a e coupled o G i/o , A 2A R o G s/ol , A 2B R o G s/q and A 3 R o G i/q p o eins (F edholm e al., 2005). A 2A R a e posi i ely coupled o AC/Cyclic adenosine monophospha e (cAMP) pa hway ha in u n ac i a es he p o ein kinase A (PKA) ha phos- pho yla es se e al a ge s such as he cAMP- esponsi e elemen binding p o ein (CREB) and dopamine- and cAMP- egula ed phosphop o ein (DARPP-32) (F edholm e al., 2005). In con as , A 1 R inhibi he ac i i y o he AC/cAMP/PKA pa hway. O he P1R signaling pa hways ha e been iden i ied such as he mi ogen-ac i a ed p o ein kinase (MAPK) pa hway (Haq e al., 1998; Me ighi e al., 2017). Changes in phospho- lipase C (PLC) ac i i y ollowing P1R ac i a ion ha e also been desc ibed depending on he cell ype and adenosine concen a ion (Ribei o e al., 2002). In e es ingly, a common ea u e o all P1Rs is he coupling o he Ex acellula signal- egula ed kinases 1/2 (ERK1/2) (Schul e e al., 2000). In addi ion, A 1 R ac i a ion can lead o channel egula ion by ac i a ing se e al po assium channels ia he β/γ sub- uni s o he G p o ein (Hosseinzadeh and S one, 1998; Ro e mund e al., 2018), o by inac i a ing N, P and Q- ype calcium channels (Ha wick e al., 2004). Finally, he ca boxy- e minal pa o A 2A R can bind eg- ula o y p o eins as anslin-associa ed p o ein X TRAX (Sun e al., 2006) and β-a es in (Bo o o-Escuela e al., 2011; Nagaoka e al., 2023). P1R exp ession du ing de elopmen has been shown o be ime- dependen . Pu ine gic ecep o s a e one o he i s cell su ace e- cep o s exp essed du ing de elopmen , wi h adenosine ecep o s being p esen in mid-la e emb yogenesis onwa ds (Bu ns ock and Dale, 2015). Using in si u hyb idiza ion and binding assays, i was shown ha A 1 R exp ession begins a emb yonic day 14 (E14) and hen g adually in- c eases un il E21 o s abilize a a le el ha pe sis s in o adul hood. A 1 R a e ubiqui ous in he b ain. They a e de ec ed in he hippocampus, halamus, midb ain, co ex, and ce ebellum (Ri kees, 1995; Wea e , 1996). In basal ac i i y condi ions, he weak ex acellula adenosine concen a ion p e e en ially ac i a es A 1 R because his ecep o displays a s onge a ini y o adenosine in compa ison wi h A 2A R (Lopes e al., 2002). A 1 R a e exp essed by neu ons, bu hey a e also p esen in glial cells (F edholm e al., 2005). The B ain RNA-Seq websi e om he labo a o y o B. Ba es (h ps://www.b ain naseq.o g/) allows compa ing he exp ession o A 1 R ansc ip s in he di e en cell ypes o he mouse and human ne ous sys em a he ea ly and ma u e de el- opmen al s ages. I shows ha A 1 R ansc ip s a e highes in OPCs. They a e also de ec ed a a highe le el in as ocy es, newly o med oligodend ocy es o myelina ing oligodend ocy es han in neu- ons. A 1 R is also ound in mic oglia and mac ophages bu a lowe le els han in neu ons. A 2A R, hey a e de ec ed a E14 in he s ia um (Johansson e al., 1997), wi h exp ession inc easing ma kedly a e bi h eaching adul le els a P14. A 2A R a e es ic ed o he s ia um, hippocampus, ol ac o y bulb, ce ebellum, and co ex (Do ia e al., 1996; Wea e , 1993). Consul a ion o he B ain RNA-Seq websi e (h ps://www.b ain naseq.o g/) o cell ype compa ison o A 2A R mRNAs e eals ha A 2A R ansc ip s a e de ec ed a high le el in mouse as ocy es and human e al as ocy es, as well as in neu ons, and a low le el in OPCs and mic oglia. Howe e , i s exp ession is much highe in endo helial cells han in o he b ain cell ypes. Recen ly, he adenosine signaling pa hway has been in es iga ed du ing he de elopmen al pe iod o synap ogenesis (be ween P3 and P16) in he a and mice hippocampus in i o and in i o. Fi s , a an- sien inc ease in e oked ac i i y-dependen elease o ATP and adeno- sine has been shown du ing he pe iod o synap ogenesis as compa ed o adul s age (P7 s P60) in he oden hippocampus (Gomez-Cas o e al., 2021), sugges ing a ole o his signaling pa hway in b ain de elopmen . The ec onucleo idases CD39 and CD73, wo enzymes in ol ed in ATP me abolism and adenosine p oduc ion, a e de ec ed a synapses du ing he pe iod o synap ogenesis (G ko i´ c e al., 2019). This ansien e- sicula elease and local p oduc ion o adenosine a he synapse is associa ed wi h an inc ease in he exp ession o A 2A R bu no o A 1 R in he hippocampus (Gomez-Cas o e al., 2021). This inc ease in A 2A R exp ession occu s a synapses. Using elec on mic oscopy, A 2A R was de ec ed in he de eloping (P7) hippocampus in i o in he pos synap ic neu on, ei he wi hin he pos synap ic densi y o a he pe iphe y o he synapse a symme ic GABAe gic synapses (Gomez-Cas o e al., 2021). In con as , A 2A R was homogeneously de ec ed along dend i es a P60. Using supe - esolu ion imaging in p ima y a hippocampal cul u es, a pe isynap ic accumula ion o A 2A R coincides wi h he pe iod o syn- ap ogenesis (Gomez-Cas o e al., 2021). The ecep o o ms clus e s a 30–40% o inhibi o y synapses a 14 days in i o (DIV14) sugges ing ha i is associa ed wi h a subse o inhibi o y synapses (Gomez-Cas o e al., 2021), whe e i egula es he s abiliza ion o nascen synapses (see below). Compa ed wi h A 1 R and A 2A R, A 2B R and A 3 R a e less exp essed in he b ain. They espond only o e y high concen a ions o adenosine (in he μ M ange). RNA-Seq analysis shows ha A 2B R ansc ip s a e de ec ed a a high le el in ma u e as ocy es, whe eas hey a e poo ly exp essed in e al as ocy es (h ps://www.b ain naseq.o g/). They a e also exp essed a high le el in OPCs. The exp ession o A 2B R ansc ip s is highe in as ocy es and OPCs han in neu ons, sug- ges ing a speci ic ole in hese cells. In con as , A 3 Rs a e mos ly exp essed by mic oglia du ing b ain de elopmen (Zhang e al., 2014) al hough hey ha e also been ound a lowe le el in human olgodend ocy es and in mice OPCs (h ps://www.b ain naseq.o g/). 2.3. P2 ecep o s P2Rs a e subdi ided in o iono opic P2X ecep o s and me abo- opic P2Y ecep o s, wi h ATP and ADP being hei main ligands. Each S. Rimbe e al. Neu opha macology 237 (2023) 109640 3 sub ype is exp essed in he b ain and is implica ed in many physiological unc ions including synap ic ansmission, long- e m plas ici y (LTP) and neu oglial in e ac ions (Jou dain e al., 2007; Khakh and No h, 2012; Koizumi e al., 2013). ATP signaling is ex emely b oad because o he di e en sub ypes o ecep o s ha ha e di e en sensi i i ies o ATP (nM o μ M ange) and i s e ec o s. Fo example, he kine ics and sensi i i y o P2XRs a y among he ecep o sub ypes. Each P2XR has i s own unc ional p ope ies and can be ound as homome s o he - e ome s (Khakh and No h, 2012). P2XRs open Na þ /Cl ¡ ion channels leading o he modula ion o in acellula ca ion homeos asis and he ac i a ion o a ious in acellula signaling molecules including MAP- K/ERK and p38 mi ogen-ac i a ed p o ein kinases (Khakh and No h, 2012; Weisman e al., 2006). P2X4, P2X7 ecep o mRNA a e exp essed in sub en icula zone- de i ed neu osphe es om adul mice (G imm e al., 2009; S a o d e al., 2007), and P2X7 a e exp essed by sub en icula zone and den a e gy us NSCs (Tsao e al., 2013), sugges ing a ole o hese ecep o s in pos na al neu ogenesis. P2X3 is exp essed e y ea ly du ing he de elopmen o he ne ous sys em. I is p esen in neu al c es cells in he de eloping zeb a ish emb yo (No on e al., 2000). In he a ne ous sys em, i s exp ession s a s a E11 (Cheung and Bu ns ock, 2002), and p og essi ely inc eases du ing he cou se o he de elopmen o he au onomic ne ous sys em, he b ain and spinal co d, highligh ing i s pa icipa ion in he o ma ion o senso y ne es and c anio acial mo oneu ons (Mass´ e e al., 2007; Mass´ e and Dale, 2012). P2X5 may also con ibu e o he gene a ion o mo oneu ons since i is de ec ed in he spinal co d a E9 and in mo oneu ons a E11 (Guo e al., 2013). In he de eloping b ain, mic oglial cells exp ess pu ine me abolism- ela ed enzymes (Dalmau e al., 2003) as well as P2X1 and P2X4 (Ha y, 2013). Eigh P2YR sub ypes, all GPCRs, wi h di e en pha macological and physiological p ope ies ha e been desc ibed. P2Y1R and P2Y2R a e hough o be he mos impo an PYRs in he CNS (Weisman e al., 2006). P2Y1R and P2Y2R ac i a ion leads o inosi ol isphospha e (IP3) p oduc ion and he mobiliza ion o calcium om in acellula calcium s o es. The concomi an ac i a ion o P2Y1R and P2Y2R leads o maximal calcium wa e p opaga ion in he cells exp essing bo h e- cep o s such as in as ocy es (Weisman e al., 2006). In he de eloping a b ain, he exp ession o he di e en P2Y e- cep o s a ies in ime and space, sugges ing di e en implica ions in he de elopmen o he ne ous sys em. P2Y1 and P2Y4 a e exp essed ea ly in de elopmen (be ween E11 and E18), while P2Y2 and P2Y6 appea la e (Cheung e al., 2003). Howe e , P2Y4 exp ession de- c eases a e bi h sugges ing a speci ic ole o his ecep o in he de elopmen o he p ena al b ain. P2Ys, h ough hei p esence a di e en imes o de elopmen , in he spinal co d (P2Y1), he en al ho n o he spinal co d (P2Y2 and P2Y4), he spinal mo o ne es (P2Y2), and he do sal oo ganglia (P2Y2), mus be impo an playe s in spinal co d and mo oneu on de elopmen . Fu he mo e, P2YR mRNAs a e de ec ed in sub en icula zone-de i ed neu osphe es o adul mice (G imm e al., 2009; S a o d e al., 2007). P2Y1R exp ession du ing de elopmen ma ches wi h a ole o his ecep o in neu o- genesis and neu onal mig a ion while i s exp ession dec eases du ing neu onal cell di e en ia ion (Resende e al., 2007). 3. Role in b ain de elopmen Since pu ine gic P1 and P2 ecep o s and he pu ine gic syn hesizing and deg ading molecules a e exp essed du ing emb yogenesis and pos na al de elopmen , his aises he ques ion o hei con ibu ion in b ain de elopmen . In ac , pu ines ha e been shown o be in ol ed in se e al s eps o b ain de elopmen ha we will now e iew. 3.1. Neu al s em cell p oli e a ion Neu al s em cells (NSCs) a e sel - enewing mul ipo en cells wi h he capaci y o gi e ise o neu ons, oligodend ocy es, and as ocy es ha a e p esen in he emb yonic as well as he adul CNS. In he adul oden b ain, neu ogenesis occu s in he sub en icula zone (SVZ) o he la e al en icles and he den a e gy us (DG) o he hippocampus (Al - man and Das, 1965; E iksson e al., 1998; Al a ez-Buylla and Ga cia-Ve dugo, 2002). NSCs can p oli e a e in an asymme ic and symme ic manne , hus main aining hei own pool o cells and allowing he di e en ia ion in o neu ons, as ocy es, and oligodend o- cy es o build a unc ional b ain (G¨ o z and Hu ne , 2005). Pu ines a e in ol ed in di e en s eps o neu ogenesis wi h i s e ec s depending on he ecep o in ol ed. Conce ning P1Rs, A 1 R was desc ibed o be he mos exp essed ecep o in adul NSCs o he SVZ and i s ac i a ion p omo es p oli e a ion in i o (Migi a e al., 2008). Impo an ly, A 1 R ac i a ion has been shown o inhibi SVZ neu ogenesis and s imula e as oglio- genesis bo h in i o and in i o (Beni o-Mu˜ noz e al., 2016). Rega ding A 2A R, i s in i o blockade has no impac on SVZ cell p oli e a ion du ing emb yogenesis (Alçada-Mo ais e al., 2021). In i o s udies ha e sug- ges ed ha ac i a ion o A 1 R o A 2A R p omo es neu al p ogeni o cell p oli e a ion (L e al., 2018). In e es ingly, in adul animals unde physiological condi ions A 2A R ac i a ion inc eases he numbe o newbo n neu ons in he den a e gy us wi hou a ec ing cell p oli e a- ion bo h in i o and in i o (Ribei o e al., 2021). Mo eo e , A 2A R KO p esen cogni i e impai men s associa ed wi h a educ ion in imma u e neu oblas p oli e a ion in he hippocampus (Moscoso-Cas o e al., 2017). In e es ingly, KO mice o he equilib a i e nucleoside ans- po e ype 1 exp ess less A 2A R, ha e exace ba ed impulsi i y, and p esen an impai men in cell p oli e a ion and neu oblas de elopmen (Oli e os e al., 2017). Conce ning P2Rs, P2Y1R is one o he main ATP ecep o con ol- ling emb yonic neu ogenesis. In adial glia, P2Y1R induces calcium elease om calcium s o es h ough he IP3R signaling and he eby he elease o ATP, g ow h ac o s and neu o ansmi e s (Elias and K ieg- s ein, 2008). P2Y1R ac i a ion gene a es calcium wa es be ween adial glial cells ac oss he en i e co ex ha a e in ol ed in he p oli e a ion o neu onal p ogeni o s and hei synch oniza ion in he S-phase o he cell cycle (Weissman e al., 2004). Consis en ly, he ou g ow h index o s ia a emb yonic neu osphe es dec eases when P2Y1R is blocked meaning P2Y1R s imula es he p oli e a ion o NSCs (Scemes e al., 2003). Simila ly, cell p oli e a ion is inhibi ed in adul mice SVZ neu osphe es ea ed wi h a P2Y1R an agonis o in neu ophe es om P2Y1R KO mice (Mish a e al., 2006). P2Y1R is no only in ol ed in he p oli e a ion o emb yonic NSCs bu i also ac s as a egula o o hei di e en ia ion (Lin e al., 2007), P2Y1R is down egula ed o pe mi he cell di e en ia ion (Lin e al., 2007). P oli e a ion can also be p o- mo ed by he ac i a ion o P2X7R, and i s down egula ion leads o neu ogenesis (Glase e al., 2014). Unde pa hological condi ions, he blockade o P2X ecep o s du ing oxygen and glucose dep i a ion has been desc ibed o up egula e SVZ neu ogenesis (Ve gni e al., 2009). 3.2. Neu onal mig a ion Neu ons bo n in he en icula zone o he neu al ube mig a e o es ablish he co ical laye s and o each he igh loca ion in he b ain acco ding o hei cell a e. Rega ding P1Rs, A 2A R is in ol ed in adial and angen ial mi- g a ions (Alçada-Mo ais e al., 2021; Sil a e al., 2013). T ea ing p egnan emale mice wi h an A 2A R an agonis dec eases soma os a in GABA in e neu ons numbe in he hippocampus a P6, whe eas he numbe o soma os a in in e neu ons is simila o ha o con ol o sp ing a adul s age indica ing a delay in neu onal angen ial mig a ion (Sil a e al., 2013). A 2A R also con ols he mig a ion o co ical p ojec ion neu ons, as using sho hai pin (sh)A 2A R in E14 mice lead o an accumula ion o mig a o y neu ons a he lowe in e media e zone (IZ) egion (Alçada-Mo ais e al., 2021). In e es ingly A 2A R ac i- a ion s imula es cAMP p oduc ion and he e is e idence o an S. Rimbe e al. Neu opha macology 237 (2023) 109640 4 implica ion o cAMP/PKA pa hway in neu onal mig a ion (S ou le e al., 2020). To ou knowledge, A 1 R, A 2B R o A 3 R implica ion in neu onal mig a ion has no been demons a ed so a . Rega ding P2Rs, P2Y1R con ols neu onal mig a ion (Lin e al., 2007; Liu e al., 2008; Scemes e al., 2003). P2Y1R is exp essed in cells o he en icula zone o he SVZ (Liu e al., 2008). Adding P2Y1R selec- i e an agonis on mice s ia al emb yonic neu osphe es dec eases adial mig a ion o neu oblas s (Scemes e al., 2003) and P2Y1R knock down a E14 in e e es wi h he mig a ion o neu onal p ogeni o s o he SVZ (Liu e al., 2008). P2Y1R ac s by p opaga ing calcium wa es in cells o he en icula and SVZ (Liu e al., 2008). 3.3. Neu onal and glial di e en ia ion Neu al s em cell di ision du ing b ain de elopmen main ains a pool o undi e en ia ed NSCs and gene a es p ogeni o s ha will di e en- ia e in o neu ons, as ocy es, o oligodend ocy es (G¨ o z and Hu ne , 2005). Pu ines a e in ol ed in all s ages o NSC di e en ia ion, whe he in he g ow h o neu onal ex ensions o in he p oduc ion o as ocy es and oligodend ocy es. Following di e en ia ion, neu al p ogeni o s unde go axonal elonga ion and dend i ic b anching. Pu ines play a ole in neu i e ou g ow h a emb yonic and ea ly pos na al s ages. Rega ding P1Rs, A 2A R enhances axonal elonga- ion h ough a mechanism independen o b ain-de i ed neu o- ophic ac o (BDNF) (Ribei o e al., 2016) al hough i is essen ial o BDNF-media ed neu onal di e en ia ion (Alçada-Mo ais e al., 2021) ia PKA ac i i y. Indeed, A 2A R ac i a ion leads o opomyosin ela ed kinase B (T kB) ecep o ansac i a ion (Lee and Chao, 2001), and BDNF can be p oduced upon A 2A R-dependen AC/cAMP/PKA s imula- ion (Jeon e al., 2011). Inhibi ion o his kinase in neu oblas oma cells inhibi s neu i ogenesis induced by A 2A R agonis (Canals e al., 2005). Al hough he ole o A 2A R in his p ocess is clea , indings a e con a- dic o y ega ding A 1 R. A 1 R agonis in an in i o model o cance cells induces neu i e ou g ow h (Canals e al., 2005). In con as , A 1 R has an inhibi o y ac ion on neu i e ou g ow h in p ima y cul u es o hippocampal neu ons. Neu ons ea ed wi h N6-Cyclopen yladenosine (CPA), a selec i e agonis o A 1 R, shows a dec ease in axonal leng h (The anan he e al., 2001). A 1 R exe s i s ac ion h ough Ras homol- ogy amily membe A (RhoA also known as Rho-associa ed, coiled-coil con aining p o ein kinase ROCK) ac i a ion since a pha macological inhibi ion o Rho kinase p e en s CPA-media ed inhibi ion o neu i e g ow h (The anan he e al., 2001). RhoA modula es cy oskele al ac in ilamen s and axon elonga ion (S e n e al., 2021). The ac i i y o he p o ein RhoA is egula ed by Rho kinase (Ka oh e al., 1998). This may be ela ed o in acellula cAMP le els as Rho kinase is inhibi ed in condi ions o high cAMP concen a ion (Akakpo e al., 2017). A 1 R is a G i -coupled GPCR ha inhibi s he ac i i y o AC and dec eases cAMP le els. This may in u n ac i a es Rho kinase leading o cy oskele al e ac ion and inhibi ion o axon elonga ion. A 2B Rs a e also in ol ed in axonal ou g ow h in he CNS. Thei ac i a ion p omo es in a- cellula cAMP le el and ne in1, a chemoa ac an signal o axon, in ol ed in neu onal di e en ia ion in do sal spinal co d explan s om E11 a emb yo (Co se e al., 2000). A 3 R has no been implica ed in his b ain de elopmen s ep. Conce ning P2Rs, P2Y1R inhibi s di e en ia ion, as i mus be down egula ed o pe mi he di e en ia ion o emb yonic neu al p e- cu so s (Lin e al., 2007). In addi ion, P2X7R p omo es p oli e a ion and main enance o undi e en ia ed s a es (Glase e al., 2014; Yuahasi e al., 2012). Impo an ly, P2Y1R posi i ely while P2X7R and P2Y13R nega i ely egula e axonal ou g ow h (del Pue o e al., 2012a; Díaz-He nandez e al., 2008a,b). Adding ATP o p ima y cul u es o mouse hippocampal neu ons dec eases axonal leng h. This equi es an inc ease in in acellula calcium le els in axons ia he ac i a ion o ligand-ga ed ca ionic channels. Con e sely, P2X7R knockdown in neu- ons using shRNA app oach inc eases he axonal leng h o neu ons a 3 days in i o (3 DIV) (Díaz-He nandez e al., 2008a,b). On he same line, neu ons ans ec ed wi h a shRNA agains P2Y13R de elop axons almos wice longe compa ed o con ol neu ons (del Pue o e al., 2012b). On he con a y, he posi i e e ec o ADP on axonal leng h is p e en ed in shP2Y1R ans ec ed neu ons (del Pue o e al., 2012b). Mo eo e , an inc ease in local cAMP le el induces axonal g ow h (Ba y e al., 2017; Shelly e al., 2010). This inc ease has been linked o P2X7, P2Y13 and P2Y1 ac i a ion and he capaci y o hese ecep o s o inhibi o ac i a e adenylyl cyclase 5 (AC5). Indeed, hese ecep o s egula e in acellula cAMP le el h ough di e en ways: P2Y13R is coupled o a G i p o ein able o inhibi AC5, and P2X7R ac i a ion leads o an in acellula calcium ele a ion also able o inhibi AC5. Con e sely, P2Y1R is coupled o G q ha ac i a es AC5 (del Pue o e al., 2012b). Di e en ia ion o NSCs can also d i e as ocy e and oligodend ocy e p oduc ion, i.e., espec i ely as ogliogenesis and oligodend o- genesis. E idence o he in ol emen o P1Rs in as ogliogenesis comes om he obse ed dec ease in as ocy e densi y in he s ia um and hippocampus o pup daily i.p. injec ed wi h ca eine, a non- compe i i e A 2A R and A 1 R an agonis , be ween P3 and P10 (Des e e e al., 2007). This e ec is ansien because i is obse ed a P7, P10 and P15 bu no a P20 and P40 i.e., a e he pe iod o as ocy e p oli e a- ion and ma u a ion (Des e e e al., 2007). A 1 R is no in ol ed in as ogliogenesis because a ea men wi h i s agonis CPA o an agonis 8-(p-sul ophenyl) 8-cyclopen yl-1,3-dip opylxan hine (DPCPX) does no modi y as ocy ic densi y. In con as , A 2A R is in ol ed since he ea men wi h a selec i e A 2A R agonis (CGS21680) inhibi s he ca eine-induced loss o as ocy es (Des e e e al., 2007). This e ec o ca eine on as ocy e densi y could be a consequence o educed NSC p oli e a ion, as A 1 R and A 2A R p omo e sel - enewal o adul NSC (Migi a e al., 2008; Ribei o e al., 2021). Wi h ega d o A 3 R, i has been shown in p ima y a cul u es a P7 ha he ecep o in luences as ocy e iabili y as a unc ion o adenosine concen a ion, ia egu- la ion o he le el o he chemokine CCL2, which is p o ec i e o apop o ic (Abb acchio e al., 1998; Wi endo p e al., 2004). Pu ines a e also in ol ed in oligodend ogenesis. Opposi e o ATP and he ac i a ion o ce ain ATP ecep o s, adenosine ea men inhibi s he p oli e a ion and p omo es he ma u a ion o OPCs. T ea ing a cul u es o OPCs wi h he po en non-selec i e adenosine A 1 , A 2A , A 2B and A 3 ecep o agonis , 5 ′ -N-e hylca boxamido adenosine (NECA), in he p esence o he mi ogen pla ele -de i ed g ow h ac o (PDGF), inc eases he p opo ion o OPCs exp essing O4 and O1 ma u- a ion ma ke s (S e ens e al., 2002). Mo eo e , P1R ac i a ion p o- mo es myelina ion in he same model bu he ecep o s in ol ed and he unde lying mechanisms a e unclea (S e ens e al., 2002). Howe e , neona al a s ea ed wi h A 1 R agonis s p esen a educ ion in whi e and g ay ma e olume and en iculomegaly (Tu ne e al., 2002). Fu he mo e, A 2A R inhibi s he di e en ia ion o oligodend ocy es in p ima y cul u es p epa ed om P1 a s, inhibi ing K + cu en (I K ) an essen ial cu en o OPC di e en ia ion (Coppi e al., 2013). 3.4. Synap ogenesis A e axon elonga ion, he neu on inds i s a ge o es ablish syn- ap ic connec ions. Synap ogenesis co esponds o he s eps o synapse o ma ion be ween neu ons in he cen al ne ous sys em o be ween neu on and (skele al/smoo h/ca diac) muscle in he pe iphe al ne - ous sys em e.g. a he neu omuscula junc ion (NMJ). Once o med, ac i e synapses eleasing neu o ansmi e s a e s abilized while he inac i e ones a e apidly des abilized and elimina ed. Since NMJs a e la ge and mo e accessible han cen al synapses, he mechanisms o synapse o ma ion and elimina ion ha e been i s s udied and unde - s ood o he NMJ. Howe e , neu on-neu on and neu on-muscle syn- ap ogenesis sha e simila mechanisms, which a e la gely egula ed by pu ines (see below). 3.4.1. Neu omuscula junc ion Ini ially, he imma u e NMJ is inne a ed by wo o mo e axons. S. Rimbe e al. Neu opha macology 237 (2023) 109640 5 Subsequen ly, he axons compe e o lea e a mono-inne a ed NMJ (Balice-Go don and Lich man, 1993; Kelle -Peck e al., 2001; Red e n, 1970). The p ocess o axon elimina ion occu s wi hin wo weeks o bi h in oden s. Thus, a he ma u e NMJ, a muscle ibe is inne a ed by only one axon, o ming a muscle uni . The neu o ansmi e eleased by he mo oneu on ending a he NMJ is ace ylcholine (ACh). I has se e al ecep o s including nico inic ecep o s (nAChR) mos ly pos synap ic and p esynap ic musca inic M1, M2, M3 and M4 ecep o s (mAChR). The ini ial s eps o synapse o ma ion a he NMJ in ol e he pos - synap ic ec ui men o nAChRs wi h he agg ega ion o exis ing e- cep o s. Then, he ma u a ion o he synapse occu s h ough an inc ease in he densi y o pos synap ic nAChRs by local syn hesis o ecep o s and a modi ica ion in ecep o subuni composi ion (B uneau and Akaa- boune, 2006; Missias e al., 1996). NMJ o ma ion is egula ed by elec ical ac i i y: blocking o ol age-dependen Na + channels, esponsible o he gene a ion o ac ion po en ials, by e odo oxin (TTX) in a scia ic ne es a P9 (Thompson e al., 1979), o inhibi ion o pos synap ic nAchRs in he soleus muscle by bunga o oxin a P5 (Dux- son, 1982) leads o poly-inne a ed NMJs. Neu onal mAChRs allow an au o- egula ion o he synapse upon p esynap ic elease o Ach: hey inhibi o s imula e ACh elease depending on he ype o mAChR ac i a ed and con ol axonal compe i ion (Nadal e al., 2016a). ATP is co- eleased wi h he neu o ansmi e ACh a he axon e minal bu also by he muscle a he NMJ (Dowdall e al., 1974; Silinsky and Hubba d, 1973). ATP p esen in he synap ic cle is implica ed in synapse ma u a ion by con olling he numbe and ype o pos - synap ic nAChRs (Fu, 1995; Jia e al., 2007). ATP s abilizes nAChRs in he memb ane o cul u ed a myo ubes (O’Malley e al., 1997). ATP is also in ol ed in ac i i y-dependen modi ica ions o he NMJ du ing de elopmen . Indeed, ATP con ols mo oneu on axon elim- ina ion by selec ing he axon ha will inne a e he muscle ibe (Fu, 1995; Jia e al., 2007). Rega ding he implica ion o P1Rs in he o ma ion o he mo o endpla e, he e minal ne es exp ess A 1 R and A 2A R, which a e mos ly de ec ed p esynap ically (Ga cia e al., 2013). The exp ession and complex in e play o hese ecep o s a he NMJ egula es ACh ans- mission du ing de elopmen o d i e he ma u a ion and axonal compe i ion. The ou P1Rs a e exp essed in mo o e minals in newbo n mice e en i he A 2A R is p edominan a ea ly de elopmen s age (Ga cia e al., 2013, 2014). Bo h A 1 R and A 2A R a e implica ed in synapse elimina ion a he NMJ du ing de elopmen . Adenosine e- cep o s ha e opposi e oles depending on he de elopmen al s age o he mouse. Blocking A 1 R wi h DPCPX o A 2A R wi h 2-(2- u - anyl)-7-(2-phenyle hyl)-7H-py azolo[4,3-e] [1, 2,4] iazolo[1,5-c]py - imidin-5-amine (SCH58261) du ing he pe iod o axonal elimina ion (P5-P15) accele a es he elimina ion a P7 meaning ha a his age adenosine signaling delays axonal elimina ion. In con as , a P9, hese p esynap ic adenosine ecep o s accele a e axonal loss o he compe i ion and p omo e pos synap ic nAChR clus e ing (Nadal e al., 2016b). This is associa ed wi h a balance be ween A 1 R and A 2A R exp essed a mo o e minals, depending on he de elopmen al s age and he amoun o adenosine p esen in he synap ic cle , which would ac i a e ei he A 1 R o A 2A R (a highe adenosine concen a ions) in o de o dec ease o inc ease, espec i ely, he synap ic elease o ACh (Co eia-de-S´ a e al., 1991; Pousinha e al., 2010; Ribei o e al., 1996). In e es ingly, some p esynap ic mAChR can po en ia e he e ec s o adenosine ecep o s on axonal compe i ion whe eas o he s ha e an agonis ic e ec (Nadal e al., 2016a). Fo example, A 1 R and mAChR M2 signaling pa hways con e ge downs eam o calcium in lux on he modula ion o AC ac i i y, wi h a nega i e c oss alk du ing in ense synap ic ac i i y o mi iga e he inhibi o y e ec o ACh elease (Oli ei a e al., 2009; Shaki zyano a e al., 2006). This unde lies he unc ional in e ac ion be ween p esynap ic ecep o s in synapse ma u a ion, as a esul o PKA and PKC pa hway balance. A p e- synap ic si e, PKA and PKC egula e calcium-dependen ACh elease (Besalduch e al., 2010; Lanuza e al., 2014; San a ´ e e al., 2009). A pos synap ic si e, PKC phospho yla es he del a subuni o he nAChR o des abilise i while, con e sely, PKA s abilizes he nAChR by phospho yla ing he epsilon subuni (Lanuza e al., 2010). In con as o A 1 R and A 2A R, he ole o A 2B R and A 3 R ha e been less s udied du ing he de elopmen o he NMJ (Ga cia e al., 2014). A 2B R is in ol ed in p omo ion o end pla e po en ial a he adul NMJ (Be na eggi e al., 2018) whe eas A 3 R inhibi s ACh elease (Cinalli e al., 2013). Conce ning P2Rs, du ing de elopmen , P2X2R exp ession pa allels he o ma ion o he NMJ (Ry en e al., 2001). A di ec in ol emen o his ecep o in he o ma ion o he NMJ was hen demons a ed in KO animals: NMJs o P2X2R–KO mice a e diso ganized wi h a misalign- men o p e- and pos synap ic elemen s and a educ ion in he densi y o olds, meaning less in agina ion o he pos synap ic pa and smalle a ea o exp ess nAChR, ha pe sis a adul s age (Ry en e al., 2007). The e o e, P2X2R egula es mo e p obably he ma u a ion o he NMJ han he ini ial s ep o o ma ion. Du ing NMJ de elopmen , ATP eleased a he synapse binds o and ac i a es p esynap ic P2 ecep o s, esul ing in a dec ease in ACh elease and hus he s abiliza ion o he mos ac i a ed syn- apse (Jia e al., 2007). A ole o p esynap ic P2Y13R in adul mice was shown, whe e P2Y13R ac i a ion, wi h i s selec i e agonis inosine 5-diphospha e sodium sal (IDP), dec eases e oked elease o ACh o ph enic ne e o s abilize he synapse (Gua acino e al., 2016). Some s udies ied o unde s and he pa hway unde lying P2Y ac i a ion leading o an inhibi ion o neu o ansmission on ma u e sys ems (Sokolo a e al., 2003). They concluded ha p esynap ic P2Y13R a e coupled wi h G i/o p o ein and ha ATP inhibi o y e ec depends on PLC and PKC. Ano he s udy epo ed he ole o p esynap ic PKC in ac i i y-dependen synapse modula ion o he NMJ (Li e al., 2004). Pe haps his mechanism could be es ablished om he de elopmen al s age. The NMJ is a ipa i e synapse composed o he mo o neu on ending, he muscle ibe and he Schwann cells ha a e in close con ac wi h he neu omuscula synapse du ing i s de elopmen (Lo e and Thompson, 1998; Oga a, 1988). Schwann cells exp ess AChR and pu ine gic ecep o s (Robi aille, 1995) and a e able o egula e he elease o neu o ansmi e s a he NMJ. Du ing de elopmen , hey sense synap ic ac i i y ia he ac i a ion o P2Y1R by ATP eleased by neu ons, which inc ease hei in acellula calcium le el ha in u n allow he elease o ATP, apidly deg aded in o adenosine in he synap ic cle . Schwann cells he e o e ein o ce s ong synapses by ac i- a ing p esynap ic A 2A R (Da abid e al., 2013, 2018). All he s eps o synapse o ma ion and ma u a ion o he NMJ in which pu ines a e in ol ed ha e been schema ized in Fig. 1. 3.4.2. Cen al synapses Al hough ATP is known o be eleased in he synap ic cle a e synap ic s imula ion in he CNS (Cunha e al., 1996; Jo and Role, 2002), i s ole a de eloping cen al synapses has been less s udied in com- pa ison wi h he NMJ. Du ing b ain de elopmen , ATP eleased om as ocy es in p e on al co ical slices om neona al a s (P3) inc eases he equency and ampli ude o spon aneous exci a o y pos synap ic cu en s (Beame e al., 2017). Howe e , he ecep o in ol ed in his egula ion was no iden i ied. Conce ning P1 adenosine ecep o s, ecen wo ks epo ed ha i egula es bo h GABAe gic and glu ama e gic synapse s abiliza ion. The adenosine signaling pa hway has been in ol ed in he s abiliza ion o nascen GABAe gic synapses in he oden hippocampus. A pha macological blockade o A 2A R des abilizes a subse o GABAe gic synapses in hippocampal cul u es, issue slices and in i o (Gomez-- Cas o e al., 2021). This ea men a ec ed he unc ionally ac i e inhibi o y synapses in i o and ex i o (Gomez-Cas o e al., 2021). Since he d ugs had a e y apid e ec (wi hin 20 min) and ha i akes se e al hou s o o m new synapses (Dobie and C aig, 2011), he adenosine signaling egula es synapse s abiliza ion a he han synapse S. Rimbe e al. Neu opha macology 237 (2023) 109640 6 o ma ion pe se. The e ec o he adenosine signaling pa hway is es ic ed o he pe iod o synap ogenesis in i o and ex i o (Gomez-- Cas o e al., 2021), highligh ing a speci ic ole du ing his key pe iod o de elopmen . Mo eo e , he exp ession in i o and in i o o a shRNA agains A 2A R in a subse o hippocampal neu ons is su icien o mimic he e ec o he ba h-applied A 2A R an agonis s on synapse numbe , indica ing ha he e ec o he d ug is speci ic, cell-au onomous and ha pos synap ic A 2A Rs a e necessa y and su icien o GABAe gic synapse s abiliza ion (Gomez-Cas o e al., 2021). I was hen p oposed ha A 2A Rs s abilize nascen GABAe gic synapses h ough he ac i- a ion o G s p o ein and calcium-Calmodulin sensi i e AC 1 and/o 8 which in u n lead o he ele a ion in in acellula cAMP and he ac i a ion o PKA. Then, PKA phospho yla es gephy in, he main sca olding p o ein a inhibi o y synapses, on a unique PKA phosphosi e (Se 305), ha in u n allows he ec ui men o iono opic GABA ype A ecep o s (GABA A R) and o he ans-synap ic o ganize s Sli K3 and PTP del a adhesion molecules (Gomez-Cas o e al., 2021) (Fig. 1). Like adenosine signaling, GABA s abilizes nascen inhibi o y synapses in he de eloping b ain (Huang and Schei ele, 2008; Oh e al., 2016; Wu e al., 2012). GABA exe s i s ac ion by inducing calcium in lux a he de eloping synapse h ough GABA A R-induced memb ane depola iza ion and he ac i a ion o ol age-dependen calcium chan- nels (Leinekugel e al., 1995; Pe o -Sinal e al., 2003). The molecula mechanism downs eam calcium s abilizing he nascen synapses emained unclea o a long ime. Recen ly, we demons a ed ha GABA A R signaling con e ge on o he adenosine signaling by ac i- a ing calcium-calmodulin, which in u n boos he ac i i y o calcium-sensi i e adenylyl cyclase AC 1/8 and he p oduc ion o cAMP in he neu on (Gomez-Cas o e al., 2021) (Fig. 1). The e o e, he AC 1/8 may ac as coinciden de ec o s o p esynap ically eleased GABA and adenosine o s abilize nascen GABAe gic synapses (Gomez-Cas o e al., 2021). In e es ingly, he ole o he adenosine signaling du ing synap o- genesis is no es ic ed o GABAe gic synapses o he hippocampus. A 2A R has been shown o be in ol ed in he p uning o glu ama e gic synapses o med be ween he e inal ganglion cells (RGC) and he do sal la e al genicula e nucleus (dLGN) du ing de elopmen (Fig. 1). T ea ing mice wi h he A 2A R an agonis 8-[(1E)-2-(2-(3,4-Dime hoxy- phenyl)e henyl]-1,3-die hyl-3,7-dihyd o-7-me hyl-1H-pu ine-2,6-dione (KW6002) in he ini ial s eps o synap ogenesis (be ween P2 and P4 and no a la e s age) enhances he seg ega ion o he e inogenicula e sys em (Miao e al., 2021), indica ing a ole o his ecep o in he e inemen o synapses du ing de elopmen . Howe e , in A 2A R KO mice in which A 2A R is absen om he ini ial s eps o b ain de elopmen , an opposi e e ec was obse ed wi h a delay in dLGN seg ega ion. The opposi e e ec s obse ed on synap ogenesis in KO mice compa ed o KW6002- ea ed animals may be explained by an impac o A 2A R dele- ion on neu ogenesis, in e neu on mig a ion, neu i e elonga ion and/o non-neu onal cell di e en ia ion and unc ion (as seen abo e) whe eas ea men o neona es be ween P2 and P4 wi h KW6002 would ha e a selec i e e ec on synap ogenesis. Miao e al. (2021) also epo ed ha he e ec o A 2A R an agonis on he seg ega ion o he e inogenicula e sys em equi es mic oglia ac i a ion, indica ing he con ibu ion o neu oglial in e ac ions in he e inemen o dLGN synapses (Fig. 1). This di e s om GABAe gic synapses in he hippocampus whe e he neu on plays a cen al ole a leas in i o since emo al o glial cells om hippocampal mixed neu on-as ocy e-mic oglia cul u es wi h ARAC did no p e en A 2A R blockade om dec easing he numbe o GABAe gic synapses du ing synap ogenesis (Gomez-Cas o e al., 2021). This sugges s ha he A 2A R is in ol ed in a mo e complex mechanism a glu ama e gic synapses han a GABAe gic synapses du ing synap ogenesis. The molecula mechanism by which A 2A R con ols glu ama e gic synap ogenesis is no ully unde s ood. Miao e al. (2021) showed ha a ansien KW6002 ea men du ing synap ogenesis educes he densi y o pos synap ic sca olding molecule Home 1 and mGluR5 wi hou al e ing he densi y o p esynap ic VGluT2 synap ic bou ons, sugges ing a pos synap ic egula ion o he synapse. Fu u e s udies will de e mine whe he Fig. 1. In ol emen o pu ine gic signaling in he o ma ion, elimina ion, and ma u a ion o pe iphe al and cen al synapses du ing de elopmen . ATP, which is eleased oge he wi h ace ylcholine (Ach) a he NMJ bu also wi h GABA and glu ama e a cen al GABAe gic and glu ama e gic synapses, is in ol ed in he key s eps o synapse o ma ion. A he de eloping NMJ, ATP ia ce ain P2YR and adenosine ia A 1 R and A 2A R, whose ac ion is egula ed by musca inic ACh ecep o s (mAChRs), a e in ol ed in he egula ion o Ach elease leading o axonal compe i ion (by elimina ing supe nume a y axons and selec ing he mos ac i e axon), and in he pos synap ic ma u a ion o he NMJ (by egula ing he ype o nAChR subuni s). The posi i e (black a ows) o nega i e ( ed lines) e ec s o ATP and adenosine signaling e lec PKC and PKA ac i i y a he p e- and pos -synap ic si es. Schwann cells can elease ATP in esponse o changes in neu onal ac i i y sensed by P2Y1R hus ein o cing s ong synapses by ac i a ing p esynap ic A 2A Rs. A hippocampal GABAe gic synapses, pos synap ic A 2A R signaling s abilizes newly o med synapses ha elease ATP and GABA by egula ing PKA ac i i y which in u n con ols pos synap ic ec ui men o GABA A Rs and he synap ogenic ans-synap ic p o eins Sli k3-PTPδ. GABA con e ges on he adenylyl cyclase pa hway o boos cAMP p oduc ion and PKA ac i a ion. A glu ama e gic synapses, pu ine gic ecep o s media e synapse elimina ion (black a ows). A 2A R signaling is in ol ed in he o ma ion o he e inogenicula e sys em by con olling he emo al o ce ain glu ama e gic synapses by a mechanism ha is s ill unknown bu equi es neu onal ac i i y and mic oglia ac i a ion. Mic oglia is also implica ed in he p uning o glu ama e gic synapses in he isual co ex ia P2Y12R ac i a ion. As ocy ic A 2B R signaling indi ec ly plays a ole in glu ama e gic synapse emodeling in he p ima y soma osenso y co ex by nega i ely egula ing he le el o mGluR5 in as ocy es which dec ease exci a o y synapse numbe . As ocy es also unes neu onal ac i i y h ough glio ansmi e elease o glu ama e and ATP a e as ocy ic P2X7R ac i a ion. S. Rimbe e al. Neu opha macology 237 (2023) 109640 7 adenylyl cyclase also unc ions as an ac i i y senso a glu ama e gic synapses o ac i a e PKA and s abilize nascen synapses as has been shown a hippocampal GABAe gic synapses, which would allow he mechanism o be gene alised o cen al synapses. On he o he hand, he s udy o he mechanisms a play a he glu ama e gic synapse should p o ide u he insigh in o he key ole o A 2A R in ela ion o mic oglia ac i a ion and con ol o synapse p uning. As ocy ic A 2B R indi ec ly plays a ole in synapse emodeling in he p ima y soma osenso y co ex by nega i ely egula ing he le el o mGluR5 in as ocy es (Tanaka e al., 2021). The e is a down egula ion o mGluR5 exp ession du ing de elopmen ha can be p e en ed by A 2B R knock down in as ocy es, hus leading o an inc ease in exci a o y synapse numbe . The e o e, as ocy ic ac i a ion o A 2B R is in ol ed in he p uning o exci a o y synapses (Fig. 1). A 1 R is known o inhibi he p obabili y o neu o ansmi e elease a adul s age (Olie and Poulain, 1999; Shen and Johnson, 1997) and seems o ha e simila unc ion a imma u e synapses. A 1 R agonis de- c eases he equency o minia u e inhibi o y pos synap ic cu en s in P12 a hippocampal slices (Jeong e al., 2003). Consis en wi h his inding, Gomez-Cas o e al. did no epo a ansien inc ease in A 1 R exp ession du ing he pe iod o synap ogenesis, as obse ed o A 2A R, and ea ing hippocampal neu ons wi h he A 1 R an agonis DPCPX did no al e GABAe gic synapse numbe . This sugges s a mino con ibu- ion o he A 1 R compa ed o A 2A R in synap ogenesis. Indeed, o he s udies showed ha he on ogenesis o A 1 R la gely sp ou s a e synapogenesis and egula es synap ic ansmission in young adul a hippocampus (Dumas and Fos e , 1998). Conce ning P2 ecep o s, hey ha e been implica ed in synapse elimina ion and egula ion. ATP eleased om p esynap ic neu ons can elici exci a o y pos synap ic cu en in pos na al a (P20) hippocampus (Pank a o e al., 1998) and co ex (Pank a o e al., 2002, 2003). A he same de elopmen al s age, as ocy ic ATP egula es he ac i i y o glu ama e gic synapses by ac i a ing p esynap ic P2Y ecep o s in hippocampal cul u es and in slices (Zhang e al., 2003). Calcium-dependen as ocy ic ATP elease s imula es P2Y1R (Yang e al., 2016). Elec ophysiology and immunos aining demons a ed ha glu ama e gic synapse elimina ion in en al pos e omedial nucleus (VPm) o he halamus is p e en ed a P16 in P2Y1R–KO mice and is no escued by ATP. In he VPm, only neu ons exp ess P2Y1R (on he con- a y o he hippocampus) (Yang e al., 2016; Zhu and Kimelbe g, 2004), meaning ha synapse elimina ion occu s h ough p e- o pos -synap ic P2Y1R ac i a ion. In he adul b ain, an in e ac ion be ween P2Y1R and he pos synap ic sca old p o ein pos synap ic densi y p o ein 95 (PSD95) ec ui s glu ama e gic ecep o s a exci - a o y synapses (Siow e al., 2010), in a o o a pos synap ic mecha- nism. Zhang e al. (2003) also shown ha adenosine om as ocy ic ATP deg ada ion dec eases he ampli ude o exci a o y pos synap ic po en ials in CA1 neu ons and pa icipa e o he e osynap ic modula ion. On he con a y, du ing b ain de elopmen , ATP eleased om as- ocy es in p e on al co ex slices om neona al (P3) a s inc eases he equency and ampli ude o spon aneous exci a o y pos - synap ic cu en s (Beame e al., 2017). Howe e , he ecep o in ol ed in his egula ion was no iden i ied. As ocy es exp ess ATP ecep o s (Fumagalli e al., 2003) and hese ecep o s a e in ol ed in synapse egula ion. ATP ecep o s allow as ocy es o sense synap ic ac i i y. As ocy ic P2X7R binds ATP eleased in he synap ic cle and ecep o ac i a ion inc eases in acellula calcium le el (Suadicani e al., 2006) ha unes neu onal ac i i y h ough glio ansmi e elease o glu ama e and ATP (A a- que e al., 1998; Pa i e al., 2001; Pas i e al., 1997). Mic oglia exp esses P2X4, P2X7, P2Y2, P2Y4, P2Y6, P2Y12, P2Y14 ecep o s (Ve kh asky e al., 2009) and all adenosine ecep o s. Mic oglia can de ec synap ic elease o ATP om neu ons and as- ocy es in pa icula h ough P2Y12R (Haynes e al., 2006). In e es - ingly, he pha macological inhibi ion o P2Y12R, which con ols ATP/ADP-dependen mic oglia chemo axis and mo ili y, p e en s neu onal ac i i y (Badimon e al., 2020). Du ing de elopmen , he mic oglial P2Y12R is implica ed in synap ic p uning o he isual co ex in he c i ical pos -na al window (Sipe e al., 2016). Ac i a ion o mic oglia by lipopolysaccha ide (LPS) induces he elease o b ain-de i ed neu o ophic ac o (BDNF) which is esponsible o mic oglial cell p oli e a ion in an A 2A R-dependen manne (Gomes e al., 2013). Howe e , he in ol emen o adenosine ecep o s in mic oglia-media ed p uning o synapses has no ye been shown. All he de elopmen al s ages in which pu ines a e in ol ed a e lis ed in Table 1. 4. Pu ine sys em de egula ion du ing de elopmen Gi en he ole o pu ine gic signaling in b ain de elopmen , i has been shown ha de egula ion in pa icula o A 1 R and A 2A R a ec he in eg i y o he b ain. A 1 R ha e been associa ed wi h b ain inju y. The pe i en icula leukomalacia (PVL) is cha ac e ized by ocal nec oses in he pe i en- icula whi e ma e leading o a en icula olume inc ease, a ea u e ha can be induced in mice by hypoxia. Impai men in oligoden- d ocy e de elopmen and axonal dis up ion may explain he whi e ma e damage (Dammann e al., 2001). In e es ingly, he en icles o A 1 R KO mice unde hypoxic condi ions a e he same size as hose o WT mice ea ed unde no moxic condi ions, indica ing ha he A 1 R plays an impo an ole in his b ain lesion (Tu ne e al., 2003). Ra s ea ed wi h A 1 R agonis CPA display educ ion in o al axonal olume, a lowe exp ession o myelin p o ein and a en iculomegaly (Tu ne e al., 2002). Howe e , i is no known whe he his is due o a loss o oligodend ocy es o a unc ional de ici . A 2A R ha e been implica ed in pa hological condi ions associa ed wi h a de ec in neu ogenesis. Oxygen and glucose dep i a ion de- c eases hippocampal p ecu so cell p oli e a ion ha can be p e en ed by pha macological blockade o A 2A R (Ma aula e al., 2013). Howe e , A 2A R blockade does no signi ican ly al e he densi y o p oli e a ing cells in he g anule cell laye in a a model o s a us epilep icus (Xu e al., 2022). Simila ly, in a mouse s ain p one o dep ession, ca eine ha non-selec i ely blocks A 1 R and A 2A R also does no al e neu on p oli e a ion in he den a e gy us (Machado e al., 2017). Impai men in A 2A R signaling a ec s he es ablishmen o unc- ional ne wo ks. The mammalian isual sys em is a de elopmen al model o synap ogenesis and neu onal plas ici y. Upon monocula enuclea ion, he e ino ec al pa hway unde goes plas ic ea angemen s ha a e es ic ed o he i s h ee pos na al weeks co esponding o he c i ical pe iod. In e es ingly, e ino ec al plas ici y is associa ed wi h up egula ion o A 1 R and A 2A R exp ession and equi es A 2A R-dependen as ocy ic eac i i y. Indeed, blocking A 2A R p e en s as ocy ic eac i i y and econnec ion o he supe io colliculus ollowing enuclea ion (Ta a es-Gomes e al., 2023). Exposing p egnan mice o ca eine o o he A 2A R selec i e an agonis KW6002 du ing he en i e de elopmen al pe iod i.e., he ges a ion and lac a ion pe iod had dele e ious consequences in he o sp ing (Sil a e al., 2013). This led o an in e neu on mig a ion de ici and a hype exci able hippocam- pus, wi h long las ing consequences such as an inc eased suscep i- bili y o seizu es and a spa ial memo y de ici (Sil a e al., 2013). Ca eine exposu e du ing he p egnancy un il P15 also a ec s he p ima y isual co ex (V1) neu ons leading o neu onal hype ac i i y in i o in his b ain egion (Fazeli e al., 2017). A blockade o A 2A R ac i i y, es ic ed o he pe iod o synap ogenesis (be ween P3 and P16) i.e., he lac a ion pe iod, by daily in ape i oneal injec ion o ca eine o a selec i e an agonis SCH58261, has simila e ec s on GABAe gic synapses and beha io as compa ed o animals ha we e ea ed h oughou de elopmen . This esul s in a 40% loss o GABAe gic synapses in he hippocampus, inc eased suscep ibili y o epilepsy and impai men in some memo y asks (no el objec loca ion bu no no el objec ecogni ion asks) (Gomez-Cas o e al., 2021). This indica es ha he e ec o A 2A R blockade h oughou S. Rimbe e al. Neu opha macology 237 (2023) 109640 8 de elopmen including du ing he ges a ion and lac a ion pe iod (Sil a e al., 2013) is p ima ily due o al e a ions in synap ogenesis. Pos - synap ic exp ession o A 2A R peaks du ing he pe iod o synap ogenesis in i o and in i o in he mouse and a co ex and hippocampus (Gomez-Cas o e al., 2021). This ansien exp ession o he ecep o coincides wi h i s ole in s abilizing GABAe gic synapses es ic ed o he pe iod o synap ogenesis. Indeed, A 2A R blockade a e he pe iod o synap ogenesis does no a ec he numbe o inhibi o y synapses (Gomez-Cas o e al., 2021). I is emp ing o p opose ha A 2A R-dependen mechanism o synapse elimina ion could be abno mally eac i a ed in pa hol- ogies whe e ex acellula adenosine inc eases o le els well abo e physiological condi ions such as in epilepsy (Dale and F enguelli, 2009), aging (Cunha e al., 1995) and Alzheime ’s disease in which A 2A R exp ession is also up egula ed (Albasanz e al., 2008; Espinosa e al., 2013). In his con ex , he massi e and con inuous o e low o ex acellula adenosine will hen o e come he es ic ed ac i a ion o A 2A Rs, leading o a p edominan ole o A 2A Rs in he de elopmen o neu odegene a ion (Ca alho e al., 2019). In line wi h his, A 2A Rs ha e been implica ed in Pa kinson’s disease and clinical ials wi h an A 2A R an agonis ha e al eady shown hei e icacy (A men e o e al., 2011; Mizuno e al., 2010). A 2A Rs ha e also been in ol ed in neu o- de elopmen al diso de s. An inc ease in A 2A R exp ession has been epo ed in spon aneously hype ensi e a s (SHR), a model o a en ion de ici hype ac i i y diso de (ADHD), and ch onic ea men wi h ca eine imp o es a en ion de ici in SHR (Pandol o e al., 2013). In F agile X synd ome, excessi e glu ama e gic signaling linked o o e - ac i a ion o he me abo opic glu ama e gic ecep o mGluR5 is a enua ed by a ch onic ea men wi h is ade ylline, an A 2A R an ag- onis , which also imp o es he associa ed lea ning de ici (Fe an e e al., 2021). Conce ning epilepsy, ATP eleased and i s u no e in o adenosine du ing seizu es con ol seizu es and he de elop- men o he disease (Don´ a e al., 2016; Lie sche e al., 2016). Seizu e-induced ATP elease om hippocampal e minals and i s me abolism in o adenosine may in u n ac i a e A 2A R on mossy ibe s esul ing in mossy ibe sp ou ing in he molecula laye o he hippo- campus in animal models o empo al lobe epilepsy as well as in pa ien s (Xu e al., 2022). Mo eo e , a local up egula ion o A 2A R in glu ama e gic synapses is obse ed in oden epilep ic models associa ed wi h an inc ease o glu ama e gic ac i i y and neu o oxici y (Canas e al., 2018). 5. Conclusions The b ain de elopmen is a highly egula ed p ocess in ol ing a mul i ude o mechanism and signaling. In his e iew, we illus a ed he ole o he pu ine gic sys em in a ious s ages o b ain de elopmen . The neona al exp ession o P1 and P2 ecep o s ha e been linked o oles o adenosine and ATP in neu ogenesis, neu onal mig a ion and di e en- ia ion as well as in synap ogenesis. The in ol emen o ATP in b ain de elopmen has been mo e ex ensi ely s udied han ha o adenosine, bu he e is g owing in e es in he ole o A 1 R and A 2A R in b ain de elopmen . A 2A R ha e a dual ole in synap ogenesis: while inhibi ion o A 2A R signaling a neona al s age al e s he de elopmen o he neu onal ne wo k, he use o A 2A R an agonis such as ca eine appea s o ha e bene icial e ec s on neu ode elopmen al and neu odegene a i e diseases. In addi ion, A 1 R and A 2A R ha e been espec i ely desc ibed as neu op o ec i e and neu odegene a i e in he adul b ain (Cunha, 2005). The e o e, unde s anding he mechanism o synapse o ma ion in ol ing pu ines du ing de elopmen should ul ima ely lead o he de elopmen o new he apeu ic a ge s o Alzheime ’s disease (Launay e al., 2023) and o he diseases in which he ATP and adenosine pa h- ways a e eac i a ed. Decla a ion o compe ing in e es The au ho s decla e no compe ing in e es s. Da a a ailabili y The au ho s do no ha e pe mission o sha e da a. Re e ences Abb acchio, M.P., Ce u i, S., B ambilla, R., Ba bie i, D., Camu i, A., F anceschi, C., Giamma ioli, A.M., Jacobson, K.A., Ca abeni, F., Malo ni, W., 1998. Adenosine A 3 ecep o s and iabili y o as ocy es. D ug De . Res. 45, 379–386. h ps://doi-o g. Table 1 Roles o pu ine gic ecep o s in b ain de elopmen . P2Y P2X A 1 A 2A A 2B A 3 Neu al s em cell p oli e a ion P2Y1 ↗ (Lin e al., 2007; Scemes e al., 2003; Weissman e al., 2004) P2X7 ↗ (Glase e al., 2014) ↗ (pos na al) (Migi a e al., 2008) ↗ (only pos na al)( Alçada-Mo ais e al., 2021; Ribei o e al., 2021) – – Neu onal mig a ion P2Y1 ↗ ( adial mig a ion) (Scemes e al., 2003; Liu e al., 2008) – – ↗ (in e neu ons) (Alçada-Mo ais e al., 2021; Sil a e al., 2013) – – Neu onal di e en ia ion P2Y1 ↘ (Lin e al., 2007) P2X7 ↘ (Glase e al., 2014; Yuahasi e al., 2012) – ↗ neu i ogenesis (Alçada-Mo ais e al., 2021; Jeon e al., 2011); Lee and Chao, 2001) – – Axonal ou g ow h P2Y1 ↗ (del Pue o e al., 2012a,b) P2Y13 ↘ (del Pue o e al., 2012b) P2X7 ↘ (Díaz-He nandez e al., 2008; del Pue o e al., 2012a) ↗ and ↘ (no clea ) ( Canals e al., 2005; The anan he e al., 2001) ↗ (Ribei o e al., 2016) ↗ (Co se e al., 2000) – As ogenesis – – – ↗ (Des e e e al., 2007) – – Oligodend ogenesis – – – ↘ di e en ia ion (Coppi e al., 2013) – – ↗ ma u a ion (S e ens e al., 2002) NMJ nAChR s abiliza ion ↗ (O’Malley e al., 1997) ↗ (Nadal e al., 2016b) ↗ (Nadal e al., 2016b) – – NMJ mono-inne a ion S abilize ac i e synapses (Jia e al.., 2007; Fu e al., 199;) ↘ axon elimina ion a P7 s ↗ a P9 (Nadal e al., 2016b) ↘ axon elimina ion a P7 s ↗ a P9 (Nadal e al., 2016b) – – GABAe gic synapse s abiliza ion – – – ↗ (Gomez-Cas o e al., 2021) – – Glu ama e gic synapse s abiliza ion P2Y1 ↘ (Yang e al., 2016) – – KO ↗ s an agonis ↘ (Miao e al., 2021) ↘ (as ocy ic) ( Tanaka e al., 2021) – S. 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