BDNF release from single cells elicits local dendritic growth in nearby neurons

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1 BDNF relese from single ells eliits lol enriti growth in nery neurons Hley Wilson Horh 1,2 n Lwrene C. Ktz 1 1 Howr Hughes Meil Institute, Deprtment of Neuroiology, Duke University Meil Center, Box 3209, Durhm, North Crolin 27710, USA 2 Current ress: Deprtments of Biology n Neurosiene, Bowoin College, 6500 College Sttion, Brunswik, Mine 04011, USA Corresponene shoul e resse to H.W.H. (hhorh@owoin.eu) Pulishe online: 30 Septemer 2002, oi: /nn927 In ulture neurons, the exogenous pplition of neurotrophins (in homogenous onentrtions) lters mny fetures of xonl n enriti rors. In vivo, however, relese of enogenous neurotrophins from neuronl proesses retes sptilly heterogeneous neurotrophin istriutions. To proe the onsequenes of suh enogenous neurotrophin istriution, we proue onor neurons in ferret ortex rin slies tht o-expresse rin-erive neurotrophi ftor (BDNF) n re fluoresent protein (RFP). Using two-photon mirosopy, we nlyze their effets on reipient neurons tht expresse green fluoresent protein (GFP) lone. BDNF relese from enrites n ell oies te iretly on nery reipient neurons to inrese enriti rnhing in istne-epenent mnner. Three-imensionl nlysis of onor n reipient enrites inite tht the BDNF soure h to e within 4.5 µm to inue enriti growth in the reipient neuron. Thus, BDNF relese from n iniviul ell lters the struture of nery enrites on n exquisitely lol sle. The evelopment of enriti rors is influene y innte geneti progrms 1, tivity 2 4 n externl moleulr ues 5 7. As severl of these moleulr ues re themselves regulte y neuronl tivity, unerstning how moleulr ues influene enriti growth n revel how tivity is trnslte into struturl hnges in the eveloping rin. BDNF, memer of the neurotrophin fmily of growth ftors, is of speil interest in this regr, s its expression n relese re moulte y neuronl tivity 8 11 n its presene n lter the struture of enrites n spines in the mmmlin CNS By virtue of its omine tivity-epenent relese n morphogeni properties, BDNF my funtion s synpti morphogen 18. To ssess BDNF s role in regulting enriti growth, however, it is neessry to present neurons with ellulr soure of BDNF tht more losely mimis physiologil onitions. To this en, we use iniviul pyrmil neurons in ortil slies s neurotrophin onors to simulte in vivo neurotrophin elivery from enogenous soures. We foun (i) tht BDNF ts s sptilly restrite signl for enriti rnhing tht is psse etween neurons, n (ii) tht it inues fol enriti growth tht my influene the funtionl properties of neuronl iruitry. RESULTS Using prtile-meite gene trnsfer, we rete two popultions of pyrmil neurons within slies of ferret visul ortex. Re onor neurons were o-trnsfete with DNA for re fluoresent protein (RFP) n n epitope-tgge BDNF onstrut, n green reipient neurons were trnsfete with GFP lone. Thirty-six hours fter trnsfetion, pirs of onor n reipient neurons in lose proximity to one nother in the x y plne were simultneously imge using two-olor, two-photon mirosopy (Fig. 1). Two-photon mirosopy not only resolve the etile struture of the reipient neurons, ut the optil setioning inherent in this metho enle us to etermine the istne etween onor n reipient enrites. We were thus le to estimte the rnge of BDNF s influene. The t presente here re se on 79 ell pirs (23 BDNF onor/reipient pirs, 29 ontrol pirs, 21 nerve growth ftor (NGF) pirs n 6 trunte TrkB (T1) ell pirs). Lol inrese in enriti rnhing We previously foun tht BDNF overexpression inreses the numer of sl enrites on onor neurons 16 (lthough the fine proesses of the resulting hlo of enrites were not lerly seen uner 960 nm light the wvelength require to simultneously visulize oth GFP n RFP). Here we foun tht the enrites of reipient neurons were ffete very ifferently, epening on their proximity to the som or enrites of onor neuron. This proximity orrelte with fol inrese in the reipient neuron s enriti rnhing (Fig. 1 ). The reipient neuron s enriti rnhes tht were lote ner BDNF onor neurons frequently grew in norml iretions or emerge from the prent enrite t unusul ngles. None of these fetures were seen in reipient enrites tht were lote ner ontrol onor neuron ell oies or enrites (ells trnsfete with RFP lone, Fig. 1e n f). These ltertions in enriti rnhing were restrite to the enrites in the immeite viinity of the onor. In ontrst to BDNF overexpression 16 or th pplition of BDNF 12, oth of nture neurosiene volume 5 no 11 novemer

2 e whih n inrese the numer of sl enrites, proximity to BDNF onor neuron left the sl enrite numer unhnge. The numer of sl enrites on reipient neurons tht were lose to (<4.5 µm) BDNF-expressing onor neurons or RFP ontrol onors ws inistinguishle (4.23 ± 0.47, n = 13 versus 4.06 ± 0.30, n = 16, t-test, P = 0.755). The hlo of enrites hrteristi of ells overexpressing BDNF ws sent 16. Furthermore, lthough BDNF overexpression in iniviul neurons erese enriti stility 16, time-lpse imging showe tht the shortterm stility of reipient enrites ws unffete y proximity to BDNF onors (n = 10, 2-hour imging, t not shown). To exmine the effets of onor lotion on the rnhing of iniviul enrites, proesses were reonstrute n the threeimensionl (3D) reltionship to the onor ws quntifie Fig. 2. BDNF-inue enriti rnhing is highly restrite n orreltes with istne from the soure of BDNF. Color-oe reonstrutions of iniviul reipient neuron enrites revel effet of istne from the soure of BDNF. From the som to the first intersetion point, the reipient enrites re renere in green. Therefter, the olor of the enrite inites the istne etween the onor neuron n reipient enrite (olore r t the ottom). Fille green retngles represent the min pil enrite, n open irles represent soms of reipient neurons. Donor proesses re epite s smll re retngles n RFP soms re fille in re for BDNF onor neurons ( e) n outline in re for ontrol onor neurons (f j). Most yellow enrites (those <4.5 µm from onor proess) re highly rnhe ( ), wheres lue n turquoise enrites (>4.5 µm from onor proess) remine unrnhe ( n e). Reonstrutions lso show fol rnhing, with no effets on enrites evient proximl to the intersetion point ( n ). In ontrst, reipient neuron enrites ner RFP ontrol ells (f j) show no inrese enriti rnhing either when lose (f j) or fr (h n j). Green irles tht represent soms re pproximtely 10 µm in imeter. f Fig. 1. Lol growth of enrites of reipient neurons in res ner BDNF onor neurons. (, ) Simultneous two-photon imging of reipient (green) n BDNF onor neurons (re) revele lol res of inrese enriti omplexity (oxes enlrge in n ). Twophoton imges onsist of stks of multiple optil setions. Reipient neurons h norml numers of sl enrites n norml overll shpe n ensity of their enriti rors. However, norml ptterns of rnhing were oserve in enrites growing in lose proximity to BDNF onor neurons, s evient in higher-power imges (, ). In ontrst, ontrol neurons expressing only RFP h no influene on GFP reipient neurons (e), even in res where reipient enrites were lose to the ontrol neuron (f). Sle rs, 10 µm. (Fig. 2). Invrily, rnhing egn to inrese preisely where the reipient neuron s enrites intersete with those of the onor neuron. Inrese rnhing ontinue istlly, ut i not propgte retrogrely to the more proximl strethes of enrite (Fig. 2 e). The proximity of the BDNF soure to the reipient enrite in 3D spe strongly influene the extent of enriti rnhing (olor-oe in Fig. 2 e). For exmple, n pil enrite of reipient ell rossing onor neuron ell oy showe ense network of rnhes when the pil enrite rosse within 4.5 µm of the ell oy (s in Fig. 2). No suh inrese rnhing ws evient when n pil enrite ws more thn 4.5 µm ove or elow the BDNF onor ell oy (s in Fig. 2e). To ontrol for the remote possiility tht inrese rnhing in reipient neurons resulte from overexpression of RFP, rther thn BDNF, we exmine the effets of ontrol neurons expressing RFP lone on the rnhing of reipient neurons. Regrless of proximity, onor neurons trnsfete with RFP lone h no influene on the rnhing of iniviul reipient neurons (Fig. 2f j). Thus, the inrese enriti rnhing in reipient neurons results from BDNF overexpression, n not RFP overexpression. Moreover, this ontrol shows tht lose proximity of e f g h i j 1178 nture neurosiene volume 5 no 11 novemer 2002

3 Fig. 3. Lol enriti growth is not generl result of neurotrophin overexpression. NGF onor neurons were rete y o-trnsfeting neurons with NGF n RFP. Even when NGF onor proesses (re) were in the sme optil setion s reipient enrites (green, ) or in jent setions (), lol enriti morphology i not iffer from ontrols. High-power imges onfirm tht reipient enrites remine simple n showe no inrese enriti rnhing ( n ). Sle rs, 10 µm. enrite of one fluoresent ell to the ell oy or enrites of nother oes not in itself inue fol enriti rnhing. To etermine whether the oserve effets of BDNF on reipient neurons were speifi to BDNF, we rete NGF onor neurons y sustituting n epitope-tgge NGF onstrut for the BDNF DNA. Unlike BDNF onors, NGF onor neurons eliite no lol or glol growth of enrites reltive to ontrols, even when the NGF onor proess n the reipient proess were within the sme optil setion (11.82 ± 0.84 µm for 16 NGF intersetion points versus ± 0.46 µm for 39 ontrol intersetion points, ANOVA, P = 0.98, Fig. 3). This inites tht the effets of BDNF on lol enrite growth were not generl effet of neurotrophin overexpression. To etermine how lose BDNF onor neuron nees to e to signifintly ffet enriti rnhing in reipient neuron, we use the numer of intervening optil setions etween onor n reipient neurons t n intersetion point (Methos). Lol enriti rnhing ner this point ws ssesse y summing the liner length of enrites within 10-µm imeter yliner entere on the intersetion point n extening ove n elow it (Fig. 4 n e). When the soure of BDNF ws within the sme optil setion (<3 µm), the verge length of reipient enrites (16.2 ± 0.88 µm, n = 39 intersetion points) ws 40% greter thn tht of RFP ontrols (11.6 ± 0.46 µm, n = 39 intersetion points), ifferene tht ws sttistilly signifint (Fig. 4i, ANOVA, P < 0.05). No inrese in enriti length ws evient when the soure of BDNF ws more thn 4.5 µm wy (Fig. 4i). As further test of whether the presene of onor proess use lol enriti sprouting in reipient neurons, for eh e f g h i j neuron we lulte the rtio etween the solute length mesurements otine ove (Fig. 4i) to mesurements otine on equivlent enrites on the opposite sie of the ror n t n equl istne from the som (Fig. 4j). A rtio of unity (1:1) inites tht there ws no ifferene, on verge, in the mount of enrite in the two loles, wheres rtio greter thn one signifies inrese rnhing ner the site of intersetion with the onor neuron. Consistent with the results from the mesurements of solute enriti length, the rtio etween the enriti lengths ner the soure of BDNF n on the opposite sie of the som ws signifintly >1 only when the soure of BDNF ws within 3 µm (Fig. 4j). Fig. 4. Quntifition of lol enriti lengths revels istneepenent, lol effets of BDNF. ( h) Exmples of metho use to quntify enriti length t lose intersetion point () n fr intersetion point (e) etween onor (re) n reipient enrite (green). In oth ses, irle ws entere on n intersetion point ientifie in the 2-D imge. The z-xis lotion of the intersetion point ws lote ( n g), n irles were lso ple in the optil setion ove ( n f) n elow ( n h), reting yliner. Denriti length ws first mesure in the entrl setion, n ny itionl enrite present ove or elow ws e. RFP n GFP proesses were lerly ololize t lose intersetions () in ontrst to fr intersetions where only the GFP enrite ws visile in iniviul optil setions (g). White irles re 10 µm in imeter. (i) Histogrm of enriti lengths inne oring to the istne etween the intersetion of the reipient n BDNF onor proesses (lk rs) n ontrol proesses (gry rs). Asterisk enotes signifint ifferene within tht in s sertine y ANOVA n then Tukey multiple omprison post-test (P < 0.05) s ompre to ontrol in eh in. Brs re men + s.e.m. (j) The rtio of the mesure enriti lengths t the point of intersetion n on the opposite sie of the enriti ror inne oring to the istne etween the intersetion of the reipient n BDNF onor neurons (lk rs) n ontrol neurons (gry rs). Asterisk enotes signifint ifferenes s in (i) (P < 0.05). Brs re men ± s.e.m. nture neurosiene volume 5 no 11 novemer

4 This inites tht signifint inreses in enriti omplexity were restrite to lotions ner the soure of BDNF. BDNF ts iretly to inue fol enriti growth As we h previously shown tht BDNF is relese from BDNFoverexpressing ells 16, we were onfient tht the fol enriti growth ws onsequene of BDNF relese from onor neurons into the extrellulr spe. The fining tht BDNF expression in one neuron eliits growth in nother oes not neessrily men, however, tht relese BDNF is the signl responsile for the enhne enriti outgrowth. The relese BDNF oul t either iretly on reipient enrites or iniretly through nery ells. To etermine whether BDNF te iretly to eliit fol enriti growth, we rete reipient neurons tht expresse ominnt-negtive TrkB reeptor, thus renering them unresponsive to BDNF. Reipient neurons were otrnsfete with GFP n n epitope-tgge form of trunte TrkB (T1) 19 lking the intrellulr signling omin 20,21. Expression of oth T1 n BDNF ws onfirme vi post-ho immunohistohemistry for ll ells (s in Fig. 5 n ), n we use only pirs of neurons in whih BDNF n T1 were expresse, respetively, for nlysis. T1-trnsfete reipient neurons i not respon to BDNF from onor neuron enrites or ell oies (Fig. 5). Their enrites were no more omplex thn ontrols, regrless of their proximity to BDNF soure. Using the sme mesurement pproh esrie ove, the verge length of ontrol reipi- Fig. 5. Overexpression of ominnt negtive form of TrkB (T1) in reipient neurons loks BDNF-inue rnhing. Reipient neurons o-trnsfete with GFP n T1 o not show inrese enriti rnhing no mtter how lose the soure of BDNF ( ). Donor n reipient proesses re in the sme optil setion in the mjority of the intersetion points in these imges. (, ) Expression of T1 n BDNF were onfirme y post-ho immunohistohemistry. () Exmple of neurons in whih my-tgge BDNF (re rrow) is expresse in the re onor neuron n FLAG-tgge T1 (green rrow) is expresse in the green reipient neuron. Sle rs, 10 µm. ent enrites lote <4.5 µm from BDNF onor proesses (omining first two ins in Fig. 3, 16.0 ± 0.69 µm, n = 59 intersetion points) ws signifintly lrger thn the verge length of T1-expressing enrites (11.0 ± 0.66 µm, n = 10 intersetion points, P = 0.005, t-test). This result onfirms the qulittive impression from the imges in Fig. 5: T1 overexpression in the reipient neuron loke BDNF-inue inreses in lol enriti growth. Therefore, BDNF from onor neurons ts on reipient neuron enrites iretly, through intertions with the TrkB reeptor. Lol BDNF oes not ffet xons or spines The effets of interellulr BDNF were most ovious on enrites, ut s BDNF n inue xon outgrowth in the CNS 15,22, we exmine whether BDNF onor neurons influene sprouting of pyrmil ell xons in reipient neurons. The min xonl trunk of pyrmil neurons frequently forms severl ollterl rnhes within few hunre mirons of the ell oy 23. Reipient xons trversing lose to soures of BDNF i not show ny rnhing (Fig. 6). In ft, exmintion of 16 intersetion points (ll within severl hunre mirons of the reipient ell oy) where the soure of BDNF ws within 4.5 µm from reipient neuron xons showe no eviene of xonl ollterl sprouting. BDNF hs lso een implite s synpti morphogen 18, so we investigte whether fol BDNF ltere the stility or morphology of iniviul spines. Consistent with previous reports of spine instility uner norml onitions 24,25, we oserve hnges in the length n shpe of ontrol filopoi n spines Fig. 6. BDNF onor neurons o not influene ollterl rnhing of reipient neuron xons. () Multiple xons trverse the hlo of BDNF onor neuron ut remine unrnhe. Sle r, 10 µm. (, ) Highpower view of iniviul xons from () tht pss within 3 mirons vertilly of BDNF onor enriti rnhes. Optil setions equivlent to 6 µm of tissue re stke in (), n 10-µm equivlent is stke in (). Sle rs (, ) re 2.5 µm nture neurosiene volume 5 no 11 novemer 2002

5 Fig. 7. Spine n filopoi hrteristis re not influene y BDNF from onor neurons. () Imges of single ontrol enriti setion over time revels tht some spines i not hnge in length (green rrowhe) n other spines hnge length rmtilly (pink rrowhe). Likewise, some filopoi ynmilly ltere their length over time (lue rrowhe), while others were muh more stle (yellow rrowhe). Sle r, 5 µm. () A grph of the hnges in spine length over time of two spines in () (pink n green rrowhes) inites lrge vrition in the mount of motility from spine to spine. () Grph of the hnges in filopoil length over time of two filopoi in () (lue n yellow). () The ensity of protrusions ws mesure t eh intersetion point n groupe y the istne etween the reipient n onor proesses. Intersetion points were ollete from 23 reipient ells (117 intersetion points) ner BDNF onor ells (lk rs) n from 29 reipient ells (159 intersetion points) ner RFP ontrol onor neurons (gry rs). There ws no effet of tretment or istne on lol ensity of spines or filopoi (ANOVA, P = 0.089). Brs re men ± s.e.m. over time (Fig. 7 ). The verge ynmis of ll protrusions on ontrol GFP-trnsfete neurons (0.52 ± 0.05 µm/10 min, n = 40 protrusions) were unhnge in the presene of fol ellulr soure of BDNF (0.46 ± 0.02 µm/10 min, n =106 protrusions, P = 0.298, t-test). Even when mesurements were limite to the spines within 5-µm rius of onor proess, there ws no BDNF influene on ynmis s ompre to spines ner RFP ontrol onors (Tle 1). We lso seprtely nlyze the motility of enriti filopoi, whih re less stle thn fully ifferentite spines. However, these too were not ifferentilly influene y fol soure of BDNF (Tle 1). Likewise, neither the length of filopoi nor tht of spines iffere from ontrols, regrless of their proximity to BDNF soure (Tle 1). Finlly, in ontrst to the effet of overexpressing BDNF in single ells, whih les to ereses in spine ensity 16, fol soure of BDNF from onor neurons i not lter the lol ensity of protrusions on reipient enrites s ompre to ontrols (Fig. 7, P =0.089, ANOVA), even when the soure of BDNF ws in the sme optil setion. DISCUSSION These results emonstrte tht BDNF supplie y single neuron influene enriti rnhing of nery enrites, with little effet on xons or spines. The inrese in enriti rnhing ws restrite to fol res of enrites, n this effet ws more shrply restrite t frther istnes from the soure of BDNF. Lol effets of BDNF on enriti rnhing The morphologil response to fol BDNF ws restrite to lol portion of the enriti rnh. There ws no inrese in rnh ensity either t the ell oy or in prts of the enriti ror fr from the soure of BDNF (Fig. 4j). These effets re reminisent of the lol effets of NGF on neurite outgrowth s ssesse in segmente ultures 26. How neurotrophins inue highly lol morphologil ltertions is unknown, espeilly in light of the ft tht lign-oun Trk reeptors re retrogrely trnsporte to the som 27,28. Reent eviene inites tht sptilly istint pplition of NGF n result in ifferentil tivtion of ownstrem pthwys 29, implying tht the funtionl result of neurotrophin pplition n e epenent on ellulr lotion. In ition, mny of BDNF s reporte effets, suh s ltering reeptor tivity 30, iretly opening ion hnnels 31 or moulting Rho GTPses 32, oul hnge the enriti ytoskeleton lolly n ontriute to the morphologil ltertions foun here. The BDNF-inue enriti rnhing ws lmost entirely ue to BDNF relese from enrites. Although BDNF ws overexpresse in these onor ells, the presene of enriti BDNF my e relisti euse oth suellulr in situ hyriiztion 33 n immunoeletron mirosopy 34 stuies hve lolize BDNF to enrites. Furthermore, these stuies show tht TrkB ololizes with BDNF in enrites of pyrmil neurons 33,34, whih provies plusile sustrte for enro-enriti BDNF signling. Wht implitions o these experiments hve for norml enro-enriti intertions? Owing to the ensity of pyrmil neurons in the ortex, there re multiple points where iniviul enrites oul influene one nother. BDNF-meite enro-enriti intertions my e espeilly importnt in other res of the rin where enrites re highly enrihe, suh s in the rrels of the somtosensory ortex or in the glomeruli of the olftory ul. The glomeruli re prtiulrly interesting euse they re extremely enrite-rih, ontining the enrites of mitrl ells, tufte ells n periglomerulr ells 35. Although we hve not iretly teste the role of neuronl tivity in the experiments reporte here, the tivity-epenent relese n response properties of BDNF 8 11,36 suggest tht enrites my respon to sptil ptterns of orrelte tivity y inuing positive feek loop tht results in the enrihment of enriti rnhes in restrite portions of enriti rors. For exmple, lol enriti rnhing ptterns seen in the stripes of three-eye frogs 4 or the retin 37 oul rely on suh BDNF-meite enriti signling uring evelopment. Constrine iffusion of BDNF Fol BDNF relese y neurons into the three-imensionl ellulr mtrix of n orgnotypi slie, omine with two-photon mirosopy, provies onsierle insight into the sptil influenes of BDNF. Anlysis of seril optil setions revele strik- nture neurosiene volume 5 no 11 novemer

6 Tle 1. BDNF i not influene spine n filopoi length or ynmis. Control BDNF Ner (<4.5 µm) Fr (>4.5 µm) Ner (<4.5 µm) Fr (>4.5 µm) Signifine Dynmis (µm/10 min) Spines 0.50 ± ± ± ± 0.05 P = 0.15 (n =) (6) (20) (59) (21) Filopoi 0.66 ± ± ± ± 0.14 P = 0.38 (n =) (3) (7) (17) (9) Length (µm) Spines 0.76 ± ± ± ± 0.16 P = 0.14 (n =) (6) (20) (59) (21) Filopoi 3.51 ± ± ± ± 0.44 P = 0.79 (n =) (3) (7) (17) (9) The ynmis or length of reipient proesses lote ner or fr from ontrol onor neurons (left) n ner or fr from BDNF onor neurons (right) re isplye. The numer of protrusions for eh tegory is note elow within prentheses. ANOVAs one on eh row of t onfirm tht BDNF i not influene spine or filopoil prmeters (fr right olumn). ing restritions in the istne over whih BDNF from onor enrites oul influene reipient enriti morphology. These results inite tht BDNF either oes not iffuse very fr, or is highly iffusile ut only effetive t high onentrtions. Eviene supports the former onlusion. With reporte isoeletri point (pi) of greter thn ten, the iffusion of BDNF is proly highly restrite s result of its intertions with the surrouning extrellulr environment 38. The high ensity of TrkB reeptors n the evelopmentl upregultion of the intive T1 form of the BDNF reeptor 39 oul sequester n internlize BDNF, further reuing oth the onentrtion n the rte of iffusion 40,41. Together these results imply tht TrkB n T1 my impee the lrey limite iffusion of BDNF, resulting in the highly lol effets of BDNF seen here. BDNF elivery vi iniviul neurons BDNF hs pleiotropi effets on neuronl morphology 42. Most experimentl protools, however, rely on th pplition of homogenous onentrtion of BDNF, whih is likely to e very ifferent from the sptil pttern of BDNF present in the rin. Unlike the lol effets presente here, th pplition or overexpression of BDNF ffet enrites glolly 12,16 n hnge the ensity of spines 14,16,43. Prmeters suh s BDNF onentrtion, urtion of exposure n sptil ptterns of TrkB reeptor tivtion lerly iffer mong these vrious methos n my explin the iverse morphologil effets of BDNF. Moreover, the physiologil onsequenes of BDNF on network properties 44 my e quite ifferent with glol s oppose to lol pplition. Neurons overexpressing BDNF relese BDNF into the extrellulr mtrix uner ontrol of the ell s relese mehnisms 16. Even if levels or ptterns of BDNF re somewht ifferent from those proue y ompletely enogenous relese, reting efine ellulr soure of BDNF is likely to more losely mimi the enogenous sitution thn o th pplition, injetion, loe es or implnttion of BDNF-trnsfete nonneuronl ells. Furthermore, the tions of BDNF oserve here my e more relisti thn in issoite ultures euse relese BDNF interts with the enogenous ssortment of extrellulr mtrix omponents s it iffuses. Reent experiments hve iretly emonstrte the tivity-epenent interellulr trnsfer of BDNF in issoite ultures 11, n the finings presente here provie eviene tht interellulr trnsfer within 3D mtrix of ells hs iret effet on enriti growth. But how norml is BDNF relese from trnsfete neurons? Both the mount relese n the pthwys use for relese (onstitutive versus regulte) might e ifferent from untrnsfete neurons, lthough ville eviene suggests tht the ifferenes my not e tht gret. Wheres BDNF levels in trnsfete lyer 2/3 neurons re higher thn in untrnsfete neurons in tht lyer, these levels re similr to tht seen in untrnsfete lyer 5 ells 16. Experiments tht hve emonstrte regulte relese of BDNF rely on overexpression, strongly suggesting tht the trnsfete neurons in our experiments use the regulte pthwy for relese 45. Future experiments might ress the regulte relese of BDNF from onor neurons y mnipulting tivity levels in these ulture slies n etermining if the interellulr effets of BDNF on enriti rnhing n e moulte. We i not iretly ssess the role of neuronl tivity in the tions of BDNF. In previous work 36, we hve shown tht loke of neuronl tivity in younger slies prevents the enriteenhning effets of BDNF. Although the ge of the nimls use here s soure for slies (postntl y 27, P27) ws somewht oler thn in erlier work, it seems likely tht the sme requirement hols here s well. In these slies, neurons exhiit low levels of spontneous tivity 46. We hve lso foun tht long-term loke of tivity y phrmologil gents n result in profoun ell eth in neurons t these ges (H.W.H. n L.C.K., unpu. oserv.). Thus, we elieve tht the fol enriti growth reporte here most likely requires the presene of ongoing spontneous tivity. Lol effets of BDNF on xons n spines As BDNF regultes xonl form uring evelopment 15,22,42, we ske whether lol soures of BDNF woul inue xon ollterl sprouting. BDNF oul potentilly influene ll prts of neuron, s reful suellulr loliztion stuies hve esrie homogenous istriutions for oth TrkB n T1 reeptors throughout xons, enrites n soms 47. Severl experiments hve shown, however, tht the xon, sl n pil enrites of single neuron n isply ifferentil responses to neurotrophins 12,15. Exmintion of the onor n reeptor pirs here inites tht the presene of BDNF is unlikely to regulte interstitil rnhing of xons within lyer II/III. The vrition in xonl responses to BDNF my e ue to tissue-speifi ifferenes s well s ifferenes in BDNF elivery methos. Fol relese of BDNF lso i not lter the ensity, length or motility of enriti spines n filopoi. In other experimentl ontexts, however, BDNF lters spine ensity 14,16,43, n ifferent experimentl onitions hve yiele opposing effets. Comining the permissive effet of BDNF in LTP inution 48, the hnges in spine morphology ssoite with LTP 49 n the morphogeni properties of BDNF 12,15,16, it seems likely tht BDNF oul t s synpti morphogen 18. Although fol pplition y our onor-ell mehnism oes not lter the lol prop nture neurosiene volume 5 no 11 novemer 2002

7 erties of spines, it oes not rule out the possiility tht BDNF oul t s synpti morphogen euse the enriti relese of BDNF esrie here my e signifintly ifferent from synptilly relese BDNF. In ition, spine ensity is still inresing t this ge in the ferret 50, whih oul mke lol hnges in spine ensities iffiult to etet. METHODS Slie ulture n trnsfetion. Coronl slies (400 µm thik) of P25 28 ferret (Mrshll Frms, North Rose, New York) visul ortex were prepre, ulture n trnsfete s previously esrie 16 exept tht slies were inute t 37 C for the entire experiment. Approvl for niml experiments ws given y the Duke University Institutionl Animl Cre n Usge Committee. A Helios Gene Gun (Bio-R, Herules, Cliforni) ws use to trnsfet neurons with omintions of onstruts enoing BDNF-my, NGF-my or T1-FLAG, n the fluoresent proteins egfp n sred (Clonteh, Plo Alto, Cliforni). The BDNF-my onstrut (gift of Y.-A. Bre) is inistinguishle from ntive BDNF with regr to its ility to support the survivl of hik sensory neurons in vitro (J. Klose & Y.-A. Bre, pers. omm.). The NGF-my use ws s previously esrie 16. Rt DNAs enoing the trunte TrkB reeptor (T1) ws tgge with FLAG epitope n lone into the pef-bos vetor 19 (gift of E. Cstren). Gene gun ullets were prepre using Bio- R protools. Briefly, 8 mg of 1.6-µm gol prtiles (Bio-R) were ote with 3 µg totl DNA/mg gol in the following omintions: 1.6:1 rtio of BDNF-my to RFP, n these prtiles were mixe with n equl mount of gol prtiles ote with 2 µg egfp. For T1 ontrols, the BDNF/RFP gol ws mixe with gol ote with 16:1 rtio of T1 to egfp. Alterntively, gol ote with 10 µg of RFP lone ws omine with GFP gol for BDNF ontrols, n gol ote with rtio of 2:1 NGF to RFP ws mixe with GFP gol for NGF ontrols. Two-photon lser snning mirosopy n t quisition. Cell pirs were first ientifie t 10 mgnifition using fluoresene isseting mirosope (Lei Mirosystems, Bnnokurn, Illinois). They were then visulize with ustom uilt 2-photon mirosope equippe with ul, externl photomultipliers (Hmmtsu, Brigewter, New Jersey). The light soure ws Ti:Spphire lser (Tsunmi, Spetr-Physis, Mountin View, Cliforni) pumpe y 10-W soli stte lser (Milleni X, Spetr-Physis). A 40, 0.75 NA wter immersion lens (Zeiss, Thornwoo, New York) ws use to illuminte the smple with pproximtely 20 mw of 960-nm light yieling optil setions with n pproximte epth of 3 µm. Both low-zoom (zoom, 1.5; step size, 1.5 µm) n highzoom (zoom, 5; step size 0.5 or 0.7 µm) imges were ollete. When spine motility ws investigte, 3 10 high zoom imge stks were sequentilly ollete. As ontrol for the lol effets of BDNF, n re n equl istne from the ell oy on the opposite sie ws lso imge, n experimentl n ontrol sies were interleve. Exept for slight ontrst n rightness justments, no imge enhnement or filtering mnipultions ws one on these imges. Immunohistohemistry. For visuliztion of BDNF-my, NGF-my n T1-FLAG expression, slies were prepre for histohemistry s esrie 16. Slies were then inute with 1:1,000 mouse nti-my ntioy (PhrMingen, Sn Diego, Cliforni) or 1:750 iotinylte nti- FLAG (M2, Gio-BRL, Githersurg, Mryln) followe y 1:500 ntimouse Cy-5 (Chemion, Temeul, Cliforni) or 1:1,000 streptviin Cy-5 (Jkson ImmunoResrh Ls, West Grove, Pennsylvni). Dt nlysis. After olletion with the 2-photon mirosope, imges were poole, ssigne n lphnumeri oe n nlyze lin to onition using IPL softwre (Snlytis, Firfx, Virgini). Every existing intersetion point ws ientifie in 2-imensionl imges n nlyze. The z-xis istne etween the onor n reipient t n intersetion point ws etermine y ounting optil setion seprtion. For exmple, proesses within the sme optil setion were onsiere to e <3 µm prt, those in jent setions (fter 1.5- µm step) were µm prt, n so forth. For enriti reonstrutions, iniviul enrites (inluing ny proess longer thn 3 µm) were tre from 2- D imge stks. Reonstrutions were olore yellow for enrites in the sme or jent optil setions (<4.5 µm), turquoise for one or two intervening optil setions ( µm) or lue for three or more intervening optil setions (>7.5 µm). Lol enriti length ws quntifie s follows. The x,y oorintes of intersetion points etween re n green ells were note in 2-D imges. The optil setion ontining the green proess ws lote. A 10-µm imeter irle ws then entere on the x,y oorintes of the intersetion point in tht optil setion s well s the optil setion immeitely ove n elow, in effet reting yliner 10 µm wie n pproximtely 6 µm tll (s in Fig. 4, se on n estimte optil setion epth of 3 µm, 1.5-µm steps). The liner enrite length tht ws present in the enter irle ws mesure first, n then itionl enriti length present in the irles ove n elow were mesure n e. Any proess over 3 µm long ws inlue in this mesurement. Mesure lengths were verge n inne on the sis of z-xis istne etween onor n reipient (sertine s esrie ove). An ANOVA ws then one to exmine the effet of tretment n z-istne on enriti length. Rtiometri t ws ollete similrly on points n equl istne from the ell oy on the opposite sie. In ses where the opposite sie of the enriti ror ws lso lose to re onor neuron, or when no opposite sie ws ville, the verge length of ll mesure opposite sites ws use for the rtio lultion. ANOVA ws performe on the rtio mesurements to ssess ftor intertion. Spines n filopoi tht were within these yliners were further nlyze. The numer of protrusions ws ounte n the ensity per 10 µm of enrite lulte. IPL ws use to mesure protrusion length in 2D imges t eh time point ollete. Lengths in Tle 1 re the verge length over time. ANOVA ws use to proe the effets of tretment n z-istne on protrusion prmeters. Aknowlegments We thnk R. Irving for tehnil ssistne n D. Lo for inisive, helpful isussions. L.C.K. is n investigtor in the Howr Hughes Meil Institute. 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