A rapid switch in sympathetic neurotransmitter release properties mediated by the p75 receptor 2002 Nature Publishing Group

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1 A rpid switch in sympthetic neurotrnsmitter relese properties medited y the p75 receptor Bo Yng *, John D. Slonimsky * nd Susn J. Birren Deprtment of Biology, Volen Center for Complex Systems, 415 South St., M/S 008, Brndeis University, Wlthm, Msschusetts 02454, USA * The first two uthors contriuted eqully to this work. Correspondence should e ddressed to S.J.B. (irren@rndeis.edu) Pulished online: 29 April 2002, DOI: /nn853 Crdic function is modulted y norepinephrine relese from innervting sympthetic neurons. These neurons lso form excittory connections onto crdic myocytes in culture. Here we report tht rin-derived neurotrophic fctor (BDNF) ltered the neurotrnsmitter relese properties of these sympthetic neuron myocyte connections in rodent cell culture, leding to rpid shift from excittory to inhiitory cholinergic trnsmission in response to neuronl stimultion. Fifteen minutes of BDNF perfusion ws sufficient to cuse this shift to inhiitory trnsmission, indicting tht BDNF promotes preferentil relese of cetylcholine in response to neuronl stimultion. We found tht p75 / neurons did not relese cetylcholine in response to BDNF nd tht neurons overexpressing p75 showed incresed cholinergic trnsmission, indicting tht the ctions of BDNF re medited through the p75 neurotrophin receptor. Our findings indicte tht p75 is involved in modulting the relese of distinct neurotrnsmitter pools, resulting in functionl switch etween excittory nd inhiitory neurotrnsmission in individul neurons. Nerve growth fctor (NGF), BDNF, neurotrophin-3 nd neurotrophin 4/5 comprise the neurotrophin fmily of neurotrophic fctors. The effects of neurotrophins on cell function re medited through lignd-specific inding to memers of the Trk fmily of receptor tyrosine kinses nd through p75, receptor cple of inding ll memers of the neurotrophin fmily 1. Neurotrophins hve extensive effects on neuronl development nd function, s they support neuronl survivl, trigger cell deth, promote process outgrowth nd modulte synptic plsticity 2. Neurotrophins ct s short-term (cute) nd long-term (chronic) modultors of synptic ctivity in the centrl nd peripherl nervous systems 3. Studies report tht neurotrophins ct presynpticlly t developing motor synpses 4, t sympthetic neuron crdic myocyte synpses 5, in hippocmpl slices 6 nd in cultured corticl neurons 7. Neurotrophins regulte fetures of neurotrnsmitter relese such s the numer of synptic vesicles, the expression of synptic proteins, the numer of docked vesicles nd the proility of vesicle fusion In the peripherl nervous system, NGF is involved in estlishing synptic connections etween sympthetic neurons nd myocytes during development 11,12 nd cutely potentites excittory neurotrnsmission in vitro 5 vi the TrkA receptor. In culture, neontl sympthetic neurons cn relese norepinephrine, cetylcholine or oth, depending on environmentl signls Thus, the cute modultion of synptic ctivity y NGF rises the question of whether neurotrophins modulte the relese of specific neurotrnsmitters. We found tht ctivtion of the p75 receptor y BDNF tretment, p75 overexpression or ctivtion of p75 signling pthwy mrkedly incresed cholinergic trnsmission nd resulted in functionl switch to n inhiitory postsynptic response. RESULTS BDNF promotes cholinergic trnsmission In culture, sympthetic neurons formed functionl connections to crdic myocytes (Fig. 1 nd d). Functionl connections re chrcterized y the stimulus-evoked relese of neurotrnsmitter 13,17, which leds to postsynptic response tht cn e mesured s chnge in myocyte et rte during neuronl stimultion (Fig. 1) 5,18. Under these culture conditions, there ws no spontneous neuronl ctivity, which llowed us to mesure the evoked response of the myocytes. We mesured the mgnitude of the functionl postsynptic response for neuron myocyte pirs cultured for 3 dys in 5 ng/ml NGF lone (control) nd supplemented with dditionl NGF, BDNF or ciliry neurotrophic fctor (CNTF) (Fig. 1 nd ). In the control condition, neuronl stimultion led to n increse in myocyte et rte. There ws smll (not sttisticlly significnt) increse in myocyte response fter the 3-dy (chronic) tretment with 50 compred to 5 ng/ml NGF, suggesting tht ny ctions of NGF on the development of synptic connectivity were sturted t 5 ng/ml NGF. In cultures treted with 100 ng/ml BDNF, neuronl stimultion led to mrked decrese in myocyte et rte (Fig. 1 nd ). BDNF did not lter the verge seline et rte of crdic myocytes (men seline et rte in control, 32 ± 12 ets/min; BDNF, 37 ± 11 ets/min). Thus, in cultures treted for 3 dys, BDNF cused net chnge nture neuroscience volume 5 no 6 june

2 c d Fig. 1. BDNF promotes cholinergic trnsmission in sympthetic neuron myocyte cocultures. () Neontl sympthetic neurons were cultured for 3 dys with neontl ventriculr myocytes in the presence of 5 ng/ml NGF either without (Control, left) or with 100 ng/ml BDNF (right). For neuron myocyte pirs (s in d), myocyte et rte ws mesured efore, during nd fter neuronl stimultion. Dt is shown s the reltive chnge in myocyte et rte compred to the verge seline for the individul myocyte (men seline et rte for control, 17 ets/min; BDNF, 33 ets/min). Although seline myocyte et rtes vried from cell to cell, verge et rte ws not ffected y BDNF tretment (see Results). () Averged dt for control (Con) nd cultures grown in the presence of dditionl NGF (50 ng/ml), BDNF (100 ng/ml) or CNTF (10 ng/ml). To confirm tht CNTF functioned s cholinergic differentition fctor, cultures were mintined for 3 weeks with or without CNTF nd then sujected to the et rte ssy. By the 3-week time-point, CNTF hd induced functionl switch to inhiitory neurotrnsmission (men ± s.e.m., n 7 for ech condition). Cultures treted with BDNF nd 3 weeks of CNTF were significntly different from controls (*, P < 0.001). (c) Atropine (1 µm) or proprnolol (10 µm) ws included in the th solution to lock cholinergic or nordrenergic trnsmission, respectively (men ± s.e.m., n 4 for ech condition). BDNF + proprnolol ws significntly different from control (Con) + proprnolol (*, P < 0.02). (d) Neurons positioned ner eting myocytes were stimulted to elicit ction potentils t 2.5 Hz (scle r, 10 µm). ergic trnsmission ws seen. BDNF significntly enhnced this inhiitory cholinergic trnsmission (Fig. 1c). Sympthetic neurons cn pckge oth norepinephrine nd cetylcholine, nd re cple of dul relese of these trnsmitters 17. We therefore suggest tht incresed BDNF lters the neurotrnsmitter profile of these neurons to fvor relese of cetylcholine, resulting in n inhiitory functionl output. from excittory to inhiitory sympthetic neurotrnsmission. This result is in contrst to the effects of ciliry neurotrophic fctor (CNTF), known cholinergic differentition fctor for sympthetic neurons 19. Synptic trnsmission ws excittory fter 3 dys of CNTF tretment, then chnged to inhiitory trnsmission fter three-week culture period (Fig. 1). Thus, BDNF-induced chnges in neurotrnsmission re rpid compred to the ctions of CNTF nd my e medited y n independent mechnism. The BDNF-dependent, stimulus-evoked decrese in myocyte et rte ws completely olished y the cetylcholine receptor ntgonist tropine (Fig. 1c), implying n effect of BDNF on cholinergic neurotrnsmission. Atropine did not cuse significnt chnge in the level of excittory nordrenergic trnsmission in control cultures (compre Fig. 1 nd 1c), lthough it uncovered n excittory component of neurotrnsmission in BDNF-treted cultures. When proprnolol ws used to lock nordrenergic trnsmission, only low level of inhiitory cholin- Presynptic ction of BDNF Do these BDNF-induced chnges in postsynptic responses reflect chnges in the presynptic relese of neurotrnsmitters, or chnges in the myocyte response to those trnsmitters? We ddressed this question y pplying neurotrnsmitter receptor gonists directly to cocultured myocytes grown in the presence or sence of BDNF. We used picospritzer (see Methods) to pply either norepinephrine (10 µm) or the cholinergic gonist muscrine chloride (25 µm) for 25, 50 or 75 ms t pressure of 10 pounds per squre inch (p.s.i.). Mesurements of myocyte et rte showed tht the responses were within the dynmic rnge of the gonist concentrtions. BDNF did not ffect the myocyte response to norepinephrine or muscrine chloride (Fig. 2), indicting tht BDNF does not lter the postsynptic response of myocytes to either excittory or inhiitory neurotrnsmitters. These results suggest tht BDNF lters presynptic properties of sympthetic neurons. Fig. 2. BDNF did not lter the postsynptic response to neurotrnsmitter. Sympthetic neuron myocyte cocultures were grown in control medium (Con), NGF or BDNF. After 3 dys () 10 µm norepinephrine or () 25 µm muscrine chloride ws pplied to eting myocyte for different pulse durtions. Myocyte et rte ws recorded for 3 min efore nd fter the pulse. The chnge in myocyte et rte fter drug ppliction is shown for ech pulse durtion (men ± s.e.m., n = 5 for ech condition). 540 nture neuroscience volume 5 no 6 june 2002

3 Fig. 3. Acute modultion of neurotrnsmitter relese y BDNF. () Whole-cell recordings were otined from control cultures nd the chnge in myocyte et rte in response to neuronl stimultion ws mesured (Con). BDNF (100 ng/ml) ws then perfused into the dish for 15 min, the sme neuron ws stimulted nd myocyte response ws mesured gin (BDNF). After 15-min wshout of the BDNF (Wsh), the neuron ws gin stimulted nd the postsynptic response mesured. Vlues re given s men ± s.e.m., n = 7; *, cute BDNF tretment significntly different from control (P < 0.02). () The switch to inhiitory trnsmission ws locked y 1 µm tropine (men ± s.e.m., n = 7). (c) Dose response curve for the cholinergic switch induced y 15-min ppliction of BDNF (men ± s.e.m., n = 5 t ech concentrtion). Cholinergic effects of BDNF re rpid We hve previously shown tht NGF cutely (within 15 minutes) promotes nordrenergic trnsmission etween sympthetic neurons nd myocytes 5. Here, y exposing excittory connections to short pulse of BDNF nd mesuring myocyte response to neuronl stimultion, we tested the hypothesis tht BDNF rpidly modultes cholinergic relese. After the cultures were grown for three dys in the control condition, we identified neuron in close proximity to eting myocyte. We stimulted the neuron nd recorded the excittory response of the myocyte. We then perfused BDNF into the dish for 15 minutes nd stimulted the neuron gin. There ws drmtic chnge to n inhiitory postsynptic response fter the 15-minute BDNF ppliction (Fig. 3). This switch ws reversile, s shown y the prtil recovery of n excittory postsynptic response fter 15-minute wshout of the BDNF. The rpidity nd reversiility of the BDNF effect indicte tht the relese properties of the cholinergic vesicle pool cn e rpidly modulted nd tht non-trnscriptionl mechnism medites BDNF-induced chnges in neurotrnsmitter relese. Inhiition fter cute BDNF perfusion ws locked y the ddition of tropine to the th solution (Fig. 3), confirming tht BDNF cused n cute increse in cholinergic trnsmission within 15 minutes. The cute effect of BDNF ws dose dependent, with shift to predominntly inhiitory trnsmission etween 50 nd 75 ng/ml BDNF (Fig. 3c). This dose response curve is decidedly steep in the ng/ml rnge. Although the resons for this re not cler, it suggests tht fctors, possily postsynptic, other thn the totl mount of cetylcholine nd norepinephrine relesed contriute to the overll level of myocyte excittion nd inhiition. p75 regultion of cholinergic trnsmission To investigte whether BDNF cts through Trk receptors to cutely promote cholinergic relese, we included K252, n inhiitor of Trk receptor tyrosine kinses, during cute BDNF tretments. BDNF continued to promote n inhiitory postsynptic response within 15 minutes in this condition (Fig. 4). In previous studies 5 nd in prllel experiments (Fig. 4), K252 locked NGF-medited potentition of excittory synptic trnsmission, reveling n NGF-dependent, stimulus-induced inhiition of myocyte et rte. Thus, TrkA medites NGF-dependent synptic potentition, nd in the sence of Trk signling second NGF signling pthwy seems to promote inhiitory trnsmission. Together with the inility of K252 to lock BDNF-dependent inhiitory trnsmission, these dt support model for non-trk-medited rpid chnge in cholinergic relese nd suggest role for the p75 receptor in the regultion of cholinergic trnsmission. Further evidence for non Trk-medited pthwy for cholinergic regultion comes from BDNF survivl experiments. NGF c supports sympthetic neuron survivl vi ctivtion of the TrkA receptor 20. Moreover, ectopic expression of TrkB in sympthetic cells lso triggers survivl response 21. Thus, if BDNF regultes synptic function y ctivtion of ny Trk receptor, it should lso provide trophic support for the sympthetic neurons. Becuse Trk-medited neuron survivl requires lower dose of neurotrophin thn Trk-medited synptic regultion 5, we resoned tht survivl response to BDNF would indicte ctivtion of Trk-medited signling pthwy 21. In the sence of NGF, however, BDNF did not support the survivl of cultured sympthetic neurons (Fig. 4). We therefore conclude tht the ctions of BDNF on sympthetic neurons in these cultures re not medited through Trk-dependent pthwy. Furthermore, BDNF did not decrese the survivl response of sympthetic neurons in the presence of NGF, indicting tht the synptic effects of BDNF re not medited through cell deth pthwys ssocited with the p75 receptor 22. In the sence of Trk-medited pthwy, these dt suggest role for p75 in modulting cholinergic trnsmission. We therefore overexpressed humn p75 specificlly in sympthetic neurons in neuron myocyte cocultures. Overexpression of p75 protein ws confirmed y immunostining nd y co-expression of yellow fluorescent protein (YFP) mrker (Fig. 5 nd ). After 3 dys, YFP controls showed excittory neurotrnsmission nture neuroscience volume 5 no 6 june

4 to connected myocytes (Fig. 5e). In contrst, p75-overexpressing neurons showed inhiitory trnsmission, even in the sence of exogenous BDNF, indicting tht p75 is involved in the regultion of cholinergic trnsmission. As cultures expressing humn p75 contined only 5 ng/ml NGF, the switch to n inhiitory connection rised the possiility of lignd-independent signling through the overexpressed p75 receptor. Such lignd-independent signling hs een previously suggested for the intrcellulr domin of p75 (ref. 23). We therefore exmined myocyte responses following the expression of mutnt p75 (p75-105) deficient in lignd inding 24,25 (Fig. 5c nd d). We exmined eight connected neuron myocyte pirs expressing mutnt p75 in the neuron. Four of the eight showed inhiitory trnsmission upon neuronl stimultion (verge chnge in myocyte et rte, 9.6 ± 1.4 ets/min), wheres the other four showed excittory trnsmission (verge chnge in myocyte et rte, 9.8 ± 2.5 ets/min). This resulted in n verge chnge in myocyte et rte midwy etween excittion nd inhiition (Fig. 5e). Post-hoc exmintion of p immunostining in two neurons, one showing n inhiitory response nd the other n excittory response, reveled similrly high levels of p expression. Thus, though p could medite switch to inhiitory trnsmission, the effect ws not s complete s tht seen for the wild-type construct. This suggests tht overexpressed p75 my hve oth lignd-dependent nd lignd-independent components. Furthermore, the imodl distriution of excittory nd inhiitory connections is consistent with the ide suggested y the dose response curve (Fig. 3c) of shrp trnsition etween these two sttes. Further evidence for regultory role of p75 comes from nlysis of the effect of BDNF in cultures derived from p75 / mice 26. In these cultures, p75-deficient sympthetic neurons formed excittory connections to myocytes (Fig. 6), nd p75 / Fig. 4. Trk-independent effects of BDNF. () Myocytes showed n increse in et rte during neuronl stimultion in the sence or presence of K252 (200 nm). After n excittory neuron myocyte pir ws found (Stim 1), either NGF (50 ng/ml) or BDNF (100 ng/ml) ws perfused into the th in the presence of K252 for 15 min, nd the sme neuron ws stimulted gin (Stim 2). With K252 in the th, oth NGF nd BDNF produced net chnge to inhiitory trnsmission upon repeted stimultion of the neuron (men ± s.e.m., n 6 for ech condition; *, P < 0.02 s compred with mtched control). () BDNF neither supported the survivl (in the sence of NGF) nor triggered the deth (in the presence of NGF) of cultured sympthetic neurons. Survivl is expressed s the percentge of neurons counted t plting tht were live t ech time point (men ± s.e.m., n = 3). cells continued to show n evoked increse in myocyte et rte fter cute BDNF tretment, wheres wild-type controls showed inhiitory trnsmission fter the sme tretment. These dt conclusively identify p75 s regultor of cetylcholine relese from sympthetic neurons. Together with evidence implicting TrkA in the regultion of nordrenergic trnsmission 5, these results suggest tht multiple signling pthwys control the pttern of neurotrnsmitter relese in developing sympthetic neurons. A numer of signling pthwys downstrem of the p75 receptor hve een identified, including the ctivtion of sphingomyelinse, which leds to the genertion of cermide 1. We investigted the p75-medited cermide pthwy y cutely treting neuron myocyte cocultures for 15 minutes with C2-cermide, memrne-perment cermide nlog tht mimics the moiliztion of cermide following p75 ctivtion 27. A 15-minute exposure to C2-cermide resulted in stimulus-evoked inhiition in myocyte et rte (Fig. 6). In control experiments, neuronl stimultion elicited n increse in myocyte et rte fter 15-minute tretment with C2-dihydrocermide, n inctive cermide compound. The effect of the C2-cermide tretment cnnot e explined y TrkA ctivtion of the cermide pthwy ecuse cermide-induced TrkA ctivtion hs not een oserved within the 15-minute time frme used in our experiments 28. Furthermore, the effects of the cute cermide tretment recpitulted the timing of cute BDNF ppliction (Fig. 3), consistent with role for p75 in rpid chnges in synptic relese. DISCUSSION Sympthetic neurons modulte myocyte et rte through ctivity-evoked relese of neurotrnsmitter. Here we show tht cultured neontl sympthetic neurons rpidly switched etween excittory nd inhiitory neurotrnsmission in response to neurotrophins. This chnge in the functionl output of sympthetic trnsmission reflected n increse in stimulus-induced cholinergic trnsmission tht ws medited presynpticlly through the p75 receptor. Activtion of the p75 signling pthwy y BDNF tretment, p75 overexpression or ctivtion of the cermide signl trnsduction pthwy resulted in incresed cholinergic trnsmission nd ctivity-evoked inhiition of myocyte responses. BDNF did not medite chnge to synptic inhiition in cultures derived from p75 / mice, further implicting p75 in the regultion of cholinergic trnsmission. These results suggest tht cotrnsmission of norepinephrine nd cetylcholine cn e dynmiclly regulted in developing sympthetic neurons y trget-derived fctors tht modulte the relese properties of different neurotrnsmitter pools. 542 nture neuroscience volume 5 no 6 june 2002

5 Fig. 5. Overexpression of the p75 receptor induced inhiitory neurotrnsmission. (, ) A neuron cotrnsfected with YFP nd humn p75 (white rrows) overexpressed the p75 protein s detected y p75 immunofluorescence. () YFP fluorescence of the trnsfected neuron; () p75 immunorectivity in the sme neuron (white rrow). Nontrnsfected neurons in the sme field showed lower endogenous p75 expression (white rrowhed). Exposure time, 200 ms; scle r, 20 µm. (c, d) A neuron cotrnsfected with YFP nd p (white rrows). (c) YFP fluorescence; (d) p75 immunorectivity. Non-trnsfected neurons showing lower p75 immunorectivity re mrked with white rrowhed (exposure time, 500 ms). (e) Myocyte response to neuronl stimultion ws mesured for YFP vector trnsfected neurons (Control), YFP-leled neurons co-expressing wild-type humn p75 (p75) or YFP-leled neurons co-expressing the mutnt humn p Men ± s.e.m., n = 4 for control, n = 4 for p75, n = 8 for p75-105; *, P < compred with control. TrkA nd p75 ctivtion Although our dt support role for p75 in promoting cholinergic relese, previous studies hve implicted TrkA ctivtion in the potentition of nordrenergic trnsmission 5. How might ctivtion of these two receptors led to divergent downstrem effects? Trk nd p75 signling pthwys re known to interct, with p75 enhncing TrkA signling when the two receptors re co-ctivted 29,30. In the sence of Trk signling, p75 ctivtes cell deth pthwys 31, n ction which cn e locked y Trk ctivtion 32. Thus, it ppers tht p75 hs the potentil to regulte events in oth Trk-dependent nd Trk-independent wys. The recent oservtion tht proneurotrophins (the initil secreted form of neurotrophins) ct s specific, high-ffinity lignds for p75 rises further possiilities of Trk-independent p75 ctivity during the modultion of neuronl function 33. Our experiments indicte tht p75 medites specific cellulr responses in the presence of low-level TrkA ctivtion nd tht the reltive rtio of Trk nd p75 signling cn ffect the functionl output of sympthetic neurotrnsmission. This model is in ccordnce with the finding tht BDNF ctivtion of p75 shifts the functionl output of sympthetic survivl pthwys even in the presence of lowlevel TrkA ctivtion 22. In our coculture system, BDNF tretment did not led to cell deth response ut rther to locl chnges in synptic function. BDNF signling through p75 hs previously een shown to trigger the deth of sympthetic neurons, ut in condition closely resemling tht used in our experiments (5 ng/ml NGF nd c e 100 ng/ml BDNF), only smll decrese in neuronl numer is seen 22. Notly, more cell deth occurred in BDNF-treted cultures in the presence of KCl, condition in which Trk receptors re not ctivted 22. The lck of such cell deth in our cultures my lso reflect differences in culture conditions, differences in degree of p75 expression or stronger co-ctivtion of TrkA in our cultures. Thus, within rnge of TrkA nd p75 signling tht supports neuronl survivl, the lnce of signling through these receptors regultes the neurotrnsmitter profile of developing synptic connections. d Fig. 6. Activtion of p75 signling pthwys promotes inhiitory trnsmission. () After identifying connected neuron myocyte pir, the verge response of myocyte to neuronl stimultion ws mesured in cultures otined from either p75 +/+ or p75 / mice (Con). BDNF ws then dded to the th for 15 min nd the stimulus-induced myocyte response mesured gin (BDNF). BDNF did not promote cholinergic trnsmission in p75 / mouse sympthetic cocultures (p75 +/+, n = 4; p75 /, n = 6; *, P < 0.03 compred with control). () Acute ppliction of C2- cermide resulted in net chnge to inhiitory trnsmission. Neuron myocyte pirs showing n excittory connection were perfused with 20 µm C2-cermide or C2-dihydrocermide (control) for 15 min, then myocyte response to neuronl stimultion ws mesured. In some experiments, 1 µm tropine ws included to lock cholinergic trnsmission. Men ± s.e.m., n = 5 for ech condition. Responses of myocytes treted with C2-cermide were significntly different from those of controls nd those of myocytes treted with C2- cermide + tropine (P < 0.001). nture neuroscience volume 5 no 6 june

6 Regultion of co-relese of multiple neurotrnsmitters A role for p75 hs een estlished in the KCl-induced relese of dopmine from rt mesencephlic neurons 34, demonstrting tht p75, s well s Trk receptors, cn regulte neurotrnsmitter relese. In other experiments, NGF-induced relese of glutmte from cereellr grnule cells ws not locked y K252, suggesting possile further role for p75 in non-evoked relese of neurotrnsmitter 35. Here we show tht p75 signling modulted ction potentil medited synptic relese nd, moreover, regulted the differentil relese of distinct neurotrnsmitter pools within individul sympthetic neurons. The regultion of cotrnsmitter relese y different trget-derived fctors my increse the modultory complexity of sympthetic neurons, indicting tht TrkA nd p75 signling helps estlish locl ptterns of neurotrnsmitter relese. The oservtion tht neurotrophin signling through p75 preferentilly promoted the relese of cetylcholine rises the question of how cholinergic nd nordrenergic trnsmitter pools re segregted. In sympthetic neurons, cetylcholine is pckged in smll synptic vesicles, wheres norepinephrine cn e found in oth lrge dense-core vesicles nd smll synptic vesicles 14,36,37. It hs een suggested tht some vesicles might pckge cetylcholine nd norepinephrine together 38, ut nlyses of the locliztion of cholinergic nd ctecholminergic trnsporters suggest tht these trnsmitters re segregted to discrete pools of smll vesicles 39. Experiments using ltrotoxin to stimulte vesicle relese suggest tht dense-core nd smll cler vesicles my e comprtmentlized differently within synptic sites nd hve different clcium requirements for relese 40. Both Trk nd p75 ctivtion re ssocited with influx of intrcellulr clcium 41, lthough the clcium pools moilized differ for the two receptors 42. Thus, the dynmics of clcium entry my e modulted s consequence of differentil Trk nd p75 ctivtion, leding to differentil neurotrnsmitter relese. Our oservtion tht p75 signling promoted the relese of cholinergic vesicles is consistent with n effect on the moiliztion of different vesicle pools. However, further work is required to fully understnd the mechnisms underlying this specificity. These results lso hve generl implictions for understnding the regultion of cotrnsmission in the nervous system. Although it is widely ccepted tht some CNS neurons cn synthesize more thn one clssicl neurotrnsmitter 43,44, current dogm holds tht they secrete only single neurotrnsmitter 45. Arguing ginst this view, recent studies demonstrte co-relese of two clssicl neurotrnsmitters in numer of centrl systems 46 48, suggesting tht cotrnsmission my hve previously unrecognized role in the regultion of complex circuitry in the nervous system. Our work in the peripherl nervous system provides evidence for mechnism through which postsynptic fctors could regulte the differentil relese of cotrnsmitters to increse the modultory complexity of neuronl circuits. METHODS Neontl sympthetic neurons nd ventriculr myocytes. Cocultures of neontl sympthetic neurons nd crdic ventriculr myocytes were prepred nd cultured essentilly s descried 12 from neontl Simonsen white rts (Simonsen Lortories, Gilroy, Cliforni) nd p75 / or wild-type mice (Jckson Lortories, Br Hror, Mine). Animl protocols were pproved y the Brndeis University Institutionl Animl Cre nd Use Committee (IACUC). Freshly isolted sympthetic neurons (7,500 15,000 neurons) were plted with crdic myocytes (50,000 myocytes) otined from the sme nimls nd cultured for 3 4 dys or 3 weeks efore nlysis. The cells were cultured in 2 MAH food contining 5 ng/ml 2.5S NGF (Upstte Biotech, Lke Plcid, New York) s descried 5. After 1 dy in culture, 1 µm cytosine rinofurnoside (ArC; Sigm, St. Louis, Missouri) ws dded to the dishes to stop cell division. Under these conditions, ArC does not hve n effect on the survivl or function of cultured sympthetic neurons 49,50. Where indicted, the medium ws supplemented either with 100 ng/ml BDNF (gift from Regeneron Phrmceuticls, Trrytown, New York), 10 ng/ml CNTF (R&D Systems, Minnepolis, Minnesot) or 50 ng/ml NGF (finl concentrtion), or with 20 µm C2-cermide or C2-dihydrocermide (Biomol, Plymouth Meeting, Pennsylvni). For survivl ssys, neurons were plted t 10,000 cells per well in 24-well dish. Neurotrophins were dded t the time of plting. Percent survivl ws clculted y compring the numer of surviving neurons t 24, 48 nd 72 h fter plting with the numer of neurons tht were on the dish 4 6 h fter plting. Duplicte wells for ech condition were counted for minimum of three experiments per condition. Humn p75 ws expressed in cocultures using Helios Gene Gun (Bio- Rd, Hercules, Cliforni). Cells were trnsfected 1 dy fter plting either with pjpa5-cd8-yfp or pnf314-cd8-gfp (control, gift from G. Bnker), or with pjpa5-cd8-yfp nd pcmv5a, n expression vector contining humn p75 cdna (gift from M. Cho). A mutnt p75 encoding p75 protein deficient in lignd inding (p75-50, gift from M. Cho) ws sucloned into pcmv5 to generte p Expression of p75 ws detected y stining with n nti-p75 ntiody tht recognized the intrcellulr domin of oth rt nd humn p75 protein (Promeg, Mdison, Wisconsin). Mesurement of functionl postsynptic response. Cultures were visulized using n inverted Olympus IX70 microscope (Olympus, Melville, New York) with differentil interference contrst (DIC) optics. Whole-cell recordings were otined using 3 4 MΩ ptch electrodes s descried 5 from sympthetic neurons tht ppered to e connected to eting myocyte. Synptic trnsmission ws exmined y mesuring the functionl postsynptic response of myocyte to neuronl stimultion. This ws chieved y mesuring the stimulus-induced chnge in myocyte et rte 5. A seline et rte ws counted for 4 min efore the presynptic neuron ws stimulted. Then the et rte ws counted for the durtion of the 3-min stimultion. Further mnipultions nd nlyses were crried out on neuron myocyte pirs showing functionl connection 5. In some cultures, fter the initil stimultion, BDNF or cermide compounds were perfused into the dish for 15 min nd myocyte et rte ws mesured during second stimultion. BDNF ws then wshed out for 15 min nd the functionl ssy repeted. Atropine (1 µm) or proprnolol (10 µm) ws included in the th solution of some dishes to lock cholinergic or nordrenergic trnsmission respectively. In some experiments, cells were continuously perfused with 200 nm K252 (Kmiy Biomedicl, Settle, Wshington) for the length of the experiment. After connected neuron myocyte pir ws found, 50 ng/ml NGF or 100 ng/ml BDNF ws wshed into the th for 15 min, nd the stimultion protocol ws repeted. Cultures used for K252 experiments were grown in either 5 ng/ml (cute BDNF experiments) or 50 ng/ml NGF (cute NGF experiments). We hd previously estlished tht cute modultion ws not ffected y the growth concentrtion of NGF 5. Neurotrnsmitter receptor gonists were pplied to eting myocytes using Picospritzer II (Generl Vlve Corp., Est Hnover, New Jersey) nd stndrd ptch pipettes. Myocytes were exposed to either 10 µm norepinephrine (Reserch Biochemicls Interntionl, Ntick, Msschusetts) or 25 µm muscrine chloride (Sigm, St. Louis, Missouri). The gonist solutions were puffed onto the myocytes t 10 p.s.i. for 25, 50 or 75 ms. Myocyte et rte ws counted for 3 min efore nd 3 min fter the ppliction. Sttistics. Significnce ws nlyzed y Student s t-tests or ANOVA followed y post-hoc tests using SttView (Acus Concepts, Berkeley, Cliforni) softwre. Acknowledgments We thnk G. Turrigino, L. Griffith, E. Mrder nd P. Sengupt for criticl reding of the mnuscript, J. Hinterneder for helpful discussions, J. Med nd E. Nokes for technicl ssistnce nd G. Bnker, M. Cho nd B. Hempsted for help with regents. This work ws supported y grnts from the US Ntionl 544 nture neuroscience volume 5 no 6 june 2002

7 Institutes of Helth (R01 NS40168) nd the Whitehll Foundtion to S.J.B. The Pew Scholrs Progrm in the Biomedicl Sciences supported this work through Pew Scholrs Awrd to S.J.B. Competing interests sttement The uthors declre tht they hve no competing finncil interests. RECEIVED 11 MARCH; ACCEPTED 10 APRIL Kpln, D. R. & Miller, F. D. Neurotrophin signl trnsduction in the nervous system. Curr. Opin. Neuroiol. 10, (2000). 2. Hung, E. J. & Reichrdt, L. F. Neurotrophins: roles in neuronl development nd function. Annu. Rev. Neurosci. 24, (2001). 3. McAllister, A. K., Ktz, L. C. & Lo, D. C. Neurotrophins nd synptic plsticity. Annu. Rev. Neurosci. 22, (1999). 4. Lohof, A. M., Ip, N. Y. & Poo, M. Potentition of developing neuromusculr synpses y the neurotrophins NT-3 nd BDNF. Nture 363, (1993). 5. Lockhrt, S. T., Turrigino, G. G. & Birren, S. J. Nerve growth fctor modultes synptic trnsmission etween sympthetic neurons nd crdic myocytes. J. Neurosci. 17, (1997). 6. Gottschlk, W., Pozzo-Miller, L. D., Figurov, A. & Lu, B. Presynptic modultion of synptic trnsmission nd plsticity y rin-derived neurotrophic fctor in the developing hippocmpus. J. Neurosci. 18, (1998). 7. Tkei, N. et l. Brin-derived neurotrophic fctor increses the stimultionevoked relese of glutmte nd the levels of exocytosis-ssocited proteins in cultured corticl neurons from emryonic rts. J. Neurochem. 68, (1997). 8. Mrtínez, A. et l. TrkB nd TrkC signling re required for mturtion nd synptogenesis of hippocmpl connections. J. Neurosci. 18, (1998). 9. Tyler, W. J. & Pozzo-Miller, L. D. BDNF enhnces quntl neurotrnsmitter relese nd increses the numer of docked vesicles t the ctive zones of hippocmpl excittory synpses. J. Neurosci. 21, (2001). 10. Collin, C. et l. Neurotrophins ct t presynptic terminls to ctivte synpses mong cultured hippocmpl neurons. Eur. J. Neurosci. 13, (2001). 11. Korsching, S. & Thoenen, H. Nerve growth fctor in sympthetic gngli nd corresponding trget orgns of the rt: correltion with density of sympthetic innervtion. Proc. Ntl. Acd. Sci. USA 80, (1983). 12. Lockhrt, S. T., Med, J. N., Pisno, J. M., Slonimsky, J. D. & Birren, S. J. Nerve growth fctor collortes with myocyte-derived fctors to promote development of presynptic sites in cultured sympthetic neurons. J. Neuroiol. 43, (2000). 13. Furshpn, E. J., Lndis, S. C., Mtsumoto, S. G. & Potter, D. D. Synptic functions in rt sympthetic neurons in microcultures. I. Secretion of norepinephrine nd cetylcholine. J. Neurosci. 6, (1986). 14. Potter, D. D., Lndis, S. C., Mtsumoto, S. G. & Furshpn, E. J. Synptic functions in rt sympthetic neurons in microcultures. II. Adrenergic/cholinergic dul sttus nd plsticity. J. Neurosci. 6, (1986). 15. Lndis, S. C. & Keefe, D. Evidence for neurotrnsmitter plsticity in vivo: developmentl chnges in properties of cholinergic sympthetic neurons. Dev. Biol. 98, (1983). 16. Brodski, C., Schnürch, H. & Dechnt, G. Neurotrophin-3 promotes the cholinergic differentition of sympthetic neurons. Proc. Ntl. Acd. Sci. USA 97, (2000). 17. Furshpn, E. J., McLeish, P. R., O Lgue, P. H. & Potter, D. D. Chemicl trnsmission etween rt sympthetic neurons nd crdic myocytes developing in microcultures: evidence for cholinergic, drenergic, nd dulfunction neurons. Proc. Ntl. Acd. Sci. USA 73, (1976). 18. Conforti, L., Tohse, N. & Sperelkis, N. Influence of sympthetic innervtion on the memrne electricl properties of neontl rt crdiomyocytes in culture. J. Dev. Physiol. 15, (1991). 19. Sdt, S., Sendtner, M. & Rohrer, H. Ciliry neurotrophic fctor induces cholinergic differentition of rt sympthetic neurons in culture. J. Cell Biol. 108, (1989). 20. Fgn, A.M. et l. TrkA, ut not TrkC, receptors re essentil for survivl of sympthetic neurons in vivo. J. Neurosci. 16, (1996). 21. Atwl, J. K., Mssie, B., Miller, F. D. & Kpln, D. R. The TrkB-Shc site signls neuronl survivl nd locl xon growth vi MEK nd P13-kinse. Neuron 27, (2000). 22. Bmji, S. X. et l. The p75 neurotrophin receptor medites neuronl poptosis nd is essentil for nturlly occurring sympthetic neuron deth. J. Cell Biol. 140, (1998). 23. Mjdn, M. et l. Trnsgenic mice expressing the intrcellulr domin of the p75 neurotrophin receptor undergo neuronl poptosis. J. Neurosci. 17, (1997). 24. Yn, H. & Cho, M. V. Disruption of cysteine-rich repets of the p75 nerve growth fctor receptor leds to loss of lignd inding. J. Biol. Chem. 266, (1991). 25. Esposito, D. et l. The cytoplsmic nd trnsmemrne domins of the p75 nd Trk A receptors regulte high ffinity inding to nerve growth fctor. J. Biol. Chem. 276, (2001). 26. Lee, F.-F. et l. Trgeted muttion of the gene encoding the low ffinity NGF receptor p75 leds to deficits in the peripherl sensory nervous system. Cell 69, (1992). 27. Dorowsky, R. T., Werner, M. H., Cstellino, A. M., Cho, M. V. & Hnnun, Y. A. Activtion of the sphingomyelin cycle through the low ffinity neurotrophin receptor. Science 265, (1994). 28. McPhee, I. & Brker, P. A. Extended cermide exposure ctivtes the TrkA receptor y incresing receptor homodimer formtion. J. Neurochem. 72, (1999). 29. Verdi, J. M. et l. p75 LNGFR regultes Trk signl trnsduction nd NGFinduced neuronl differentition in MAH cells. Neuron 12, (1994). 30. Hntzopoulos, P. A., Suri, C., Glss, D. J., Goldfr, M. P. & Yncopoulos, G. D. The low ffinity NGF receptor, p75, cn collorte with ech of the trks to potentite functionl responses to the neurotrophins. Neuron 13, (1994). 31. Cscci-Bonnefil, P., Crter, B. D., Dorowsky, R. T. & Cho, M. V. Deth of oligodendrocytes medited y the interction of nerve growth fctor with its receptor p75. Nture 383, (1996). 32. Yoon, S. O., Cscci-Bonnefil, P., Crter, B. & Cho, M. Competitive signling etween TrkA nd p75 nerve growth fctor receptors determines cell survivl. J. Neurosci. 18, (1998). 33. Lee, R., Kermni, P., Teng, K. K. & Hempsted, B. L. Regultion of cell survivl y secreted proneurotrophins. Science 294, (2001). 34. Blöchl, A. & Sirrenerg, C. Neurotrophins stimulte the relese of dopmine from rt mesencephlic neurons vi Trk nd p75 Lntr receptors. J. Biol. Chem. 271, (1996). 35. Numkw, T., Tkei, N., Ymgishi, S., Ski, N. & Htnk, H. Neurotrophin-elicited short-term glutmte relese from cultured cereellr grnule neurons. Brin Res. 842, (1999). 36. De Potter, W. P., Prtoens, P. & Strecker, S. Nordrenline storing vesicles in sympthetic neurons nd their role in neurotrnsmitter relese: n historicl overview of controversil issues. Neurochem. Res. 22, (1997). 37. Lndis, S. C. Rt sympthetic neurons nd crdic myocytes developing in microcultures: correltion of the fine structure of endings with neurotrnsmitter function in single neurons. Proc. Ntl. Acd. Sci. USA 73, (1976). 38. Johnson, M. I., Pik, K. & Higgins, D. Rpid chnges in synptic vesicle cytochemistry fter depolriztion of cultured cholinergic sympthetic neurons. J. Cell Biol. 101, (1985). 39. Weihe, E., To-Cheng, J. H., Schfer, M. K., Erickson, J. D. & Eiden, L. E. Visuliztion of the vesiculr cetylcholine trnsporter in cholinergic nerve terminls nd its trgeting to specific popultion of smll synptic vesicles. Proc. Ntl. Acd. Sci. USA 93, (1996). 40. Sudhof, T.C. α-ltrotoxin nd its receptors: neurexins nd CIRL/ltrophilins. Annu. Rev. Neurosci. 24, (2001). 41. Stoop, R. & Poo, M. Synptic modultion y neurotrophic fctors: differentil nd synergistic effects of rin-derived neurotrophic fctor nd ciliry neurotrophic fctor. J. Neurosci. 16, (1996). 42. Jing, H. et l. Nerve growth fctor (NGF)-induced clcium influx nd intrcellulr clcium moiliztion in 3T3 cells expressing NGF receptors. J. Biol. Chem. 274, (1999). 43. Sloviter, R.S. et l. Bsl expression nd induction of glutmte decroxylse nd GABA in excittory grnule cells of the rt nd monkey hippocmpl dentte gyrus. J. Comp. Neurol. 373, (1996). 44. Gonzlez-Hernndez, T., Brroso-Chine, P., Acevedo, A., Slido, E. & Rodriguez, M. Co-locliztion of tyrosine hydroxylse nd GAD65 mrna in mesostritl neurons. Eur. J. Neurosci. 13, (2001). 45. Sulzer, D. & Ryport, S. Dle s principle nd glutmte co-relese from ventrl midrin dopmine neurons. Amino Acids 19, (2000). 46. Jons, P., Bischoferger, J. & Sndkühler, J. Co-relese of two fst neurotrnsmitters t centrl synpse. Science 281, (1998). 47. Sulzer, D. et l. Dopmine neurons mke glutmtergic synpses in vitro. J. Neurosci. 18, (1998). 48. Wlker, M. C., Ruiz, A. & Kullmnn, D. M. Monosynptic GABAergic signling from dentte to CA3 with phrmcologicl nd physiologicl profile typicl of mossy fier synpses. Neuron 29, (2001). 49. Chun, L. L. Y. & Ptterson, P. H. Role of nerve growth fctor in the development of rt sympthetic neurons in vitro III. Effect on cetylcholine production. J. Cell Biol. 75, (1977). 50. Mrtin, D. P., Wllce, T. L. & Johnson, E. M. Cytosine rinoside kills postmitotic neurons in fshion resemling trophic fctor deprivtion: evidence tht deoxycytidine-dependent process my e required for nerve growth fctor signl trnsduction. J. Neurosci. 10, (1990). nture neuroscience volume 5 no 6 june