Journal of NeurochemistryLippincott—Raven Publishers, Philadelphia© 1997 International Society for Neurochemistry

Brain-Derived Neurotrophic Factor Increases the Stimulation-Evoked Release of Glutamate and the Levels of

Exocytosis-Associated Proteins in CulturedCortical Neurons from Embryonic Rats

Nobuyuki Takei, Kumi Sasaoka, Ko Inoue, *Masamj Takahashi,Yasuhisa Endo, and tHiroshi Hatanaka

Department of Applied Biology, Kyoto Institute of Technology, Kyoto,’ *Department of Neuroscience,Mitsubishi Kasei Institute for Life Sciences, Tokyo; and fDivision of Protein Biosynthesis,

Institute for Protein Research, Osaka University, Osaka, Japan

Abstract: Differentiation and survival of neurons inducedby neurotrophins have been widely investigated, but littlehas been reported about the long-term effect of brain-derived neurotrophic factor (BDNF)on synaptic transmis-sion. Among many steps of neurotransmission, one im-portant step is regulated release of transmitters. There-fore, the release of glutamate and GABA from corticalneurons cultured for several days with or without BDNFwas measured by an HPLC-fluorescence method. Al-though BDNF had little effect on the basal release ofglutamate, high K + -evoked releasewas greatly increasedby BDNF. BDNF also tended to increase evoked releaseof GABA. Recently, several proteins involved in the stepof “regulated release” have been identified. Thus, theeffect of BDNF on the levels of these proteins was theninvestigated. Neurons were cultivated with or withoutBDNF, collected, and electrophoresed for western blot-ting. BDNF increased levels of synaptotagmin, synapto-brevin, synaptophysin, and rab3A, which were known asvesicle protein. Levels of syntaxin, SNAP-25, and /3-SNAP were also increased by BDNF. In addition, thenumbers of cored and clear vesicles in nerve terminalsor varicosities were also increased by BDNF. These re-sults raise the possibility that BDNF increases regulatedrelease of neurotransmitters through the up-regulation ofsecretory mechanisms. Key Words: Brain-derived neuro-trophic factor—Neurotrophin-3—Neurotrophin— Exo-cytosis—Synaptic vesicle—Transmitter release.J. Neurochem. 68, 370—375 (1997).

zymes, and the uptake of neurotransmitters. Amongthese factors, the functions of neurotrophins are thebest characterized. Nerve growth factor (NGF) in-creases acetyicholine (ACh) content and the activityof choline acetyltransferase (ChAT) in neurons of thebasal forebrain (Hefti et al., 1985; Hatanaka et al.,1988; Takei et al., 1988). Brain-derived neurotrophicfactor (BDNF) increases ChAT activity in septal neu-rons (Alderson et al., 1990; Knusel et al., 1991) andtyrosine hydroxylase activity in nigral neurons (Hy-man et al., 1991; Knusel et al., 1991). BDNF alsohas been reported to increase GABA uptake in striatalneurons (Mizuno et al., 1994). Neurotrophin-4/5 stim-ulates GABAergic neurons in the cortex (Widmer andHefti, 1994). In addition to these small molecular nell-rotransmitter systems, effects of BDNF on the expres-sion of neuropeptides have also been reported (Nawaet al., 1993, 1994; Croll et al., 1994; Takei et al.,1996).

Although the effects of neurotrophic factors on thecontents and synthesizing activities of neurotransmit-ters are well characterized, their effects on dynamicfunctions such as release of transmitters have been lesswell documented. We have reported previously thatregulated release of ACh from cultured basal forebrainneurons was up-regulated by NGF (Takei eta!., 1989).Recently, it was reported that severa! proteins are in-

Neuronal differentiation and maturation are thoughtto be regulated by neurotrophic factors. Many neuro-trophic factors have been identified and shown to playcrucial roles in a wide variety of neurons in the CNS.These factors have been reported to up-regulate neuro-transmitter systems such as the content of neurotrans-mitters, the activities of transmitter-synthesizing en-

Received June 7, 1996; revised manuscript received August 23,1996; accepted August 23, 1996.

Address correspondence and reprint requests to Dr. N. Takei atDepartment of Applied Biology, Kyoto Institute of Technology, Ma-tsugasaki, Sakyo, Kyoto 606, Japan.

Abbreviations used: ACh, acetylcholine; BDNF, brain-derivedneurotrophic factor; ChAT, choline acetyltransferase; GAP-43, 43-kDa growth-associated protein; MAP-2, microtubule-associated pro-tein 2; NGF, nerve growth factor; NT-3, neurotrophin-3; SNAP,soluble N-ethylmaleimide-sensitive factor attachment protein.

370

BDNF EFFECTS ON SYNAPTIC TRANSMISSION 37’

volved directly invesicle fusion and exocytosis of neu-rotransmitters (Sudhof, 1995). It is difficult to analyzethe changes in levels of these proteins in basal fore-brain owing to the small populationof NGF-responsiveneurons in this region. In contrast, BDNF- or neuro-trophin-3 (NT-3 )-responsive, i.e., TrkB- or TrkC-ex-pressing, neurons are widely distributed in the cortex(Lindsay et al., 1994). Therefore, using a culture sys-tem of embryonic rat cortical neurons, the effects ofBDNF on the release of glutamate and GABA wereinvestigated. Furthermore, the effects of BDNF andNT-3 on the levels of proteins that are associated withexocytosis were also investigated.

MATERIALS AND METHODS

MaterialsBDNF and NT-3 were kind gifts from Regeneron. Anti-

microtubule-associated protein-2 (anti-MAP-2) antiserumwas kindly supplied by Dr. H. Murofusi (University ofTokyo). Anti-43-kDa growth-associated protein (anti-GAP-43) monoclonal antibody was purchased from TDL. Anti-actin and anti-tubulin monoclonal antibodies were obtainedfrom Amersham. Anti-synaptophysin monoclonal antibody(SY38) was purchased from Boehringer Mannheim. Anti-syntaxin monoclonal antibody (lOH5) (Yoshida et a!.,1992), anti-synaptotagmin monoclonal antibody (lDI2)(Takahashi et al., 1991), and anti-synaptobrevin-2, anti-SNAP-25, and anti-/3-SNAP polyclonal antibodies (Oho etal., 1995) were prepared as reported previously. A syntheticC-terminal peptide [(C) GPQLTDQQAPPHQD I of rab3Awas conjugated to keyhole limpet hemocyanin and used toimmunize rabbits. Antiserum was purified by protein A-Sepharose and antigen affinity column chromatography.Minimum essential medium and fetal bovine serum wereobtained from GIBCO. ECL blotting reagent and PVDFmembranes were purchased from Amersham and Millipore,respectively. TheEpon 812 resin kit was obtained from Taab.All other chemicals were of reagent grade.

Cell cultureThe method of primary culture of cortical neurons was

described previously (Takei et a!., 1991; Takei and Endo,1994). In brief, cerebral cortices were removed asepticallyfrom 17-day embryonic rat fetuses. Thetissues were digestedby trypsin/DNase I and dispersed by pipetting. Dissociatedneurons were seeded onto poly-L-lysine-coated culture platesor dishes at a density of 2.5 >< i0~cells/cm2. Neurons werecultivated in minimum essential medium containing 10%fetal bovine serum. Medium was changed at day 3 in vitro,and BDNF was added to theculture at days 1 and 3 in vitro.After 5 days, cells were used for release assay or collectedfor western blot analysis or fixed for electron microscopy.

Quantification of amino acid transmittersDetermination of glutamate and GABA release from the

cultured neurons was basically the same as in our previousreport (Takei et al., 1989). In brief, neurons were washedfive times with assay buffer, HEPES-buffered Krebs—Ringersolution containing 130 mM NaC1, 5.4 mM KC1, 1.8 mMCaCI

2, 0.8 mM MgSO4, 5.5 mM glucose, and 50 mMHEPES buffer. High-K + solution containing 50 mM KC1 inKrebs—Ringer solution was prepared for depolarizing stimu-lation. All the assay buffer and culture plates were kept at

37°C.Assay buffer was changed every 3 mm and collectedinto tubes on ice. Tomake derivatives of amino acids detect-able by fluorescence monitoring, samples were mixed witho-phthalaldehyde (4:1) for 5 mm. The sample was then ap-plied to HPLC and detected by fluorescence monitoring (ex-citation at 340 nm, extinction at 445 nm).

Electrophoresis and western blot analysisCells were washed three times with TN buffer [10 mM

Tris HCI (pH 7.5) and 150 mM NaC1], scraped off thedishes using a rubber policeman, and centrifuged. The pelletwas sonicated in ice-cold TN buffer, and theprotein concen-tration was determined by the method of Bradford (1976)using bovine immunoglobulins as a standard. Samples weretreated with sodium dodecyl sulfate sample buffer (finalconcentrations, 2% sodium dodecyl sulfate, 10% glycerol,and 5% 2-mercaptoethanol in 0.0625 M Tris HC1, pH 6.8),and each cell lysate was subjected to sodium dodecyl sul-fate—polyacrylamide gel electrophoresis using 10 or 15%gels and electrophoretically transferred onto PVDF mem-branes using a semidry blotting apparatus. The polyvinyli-dene difluoride membranes were incubated in block-ing buffer [TN buffer containing 0.05% Tween 20 and 5%(wt/vol) skim milk (DIFCO)] overnight at 4°Cto blocknonspecific binding of the IgG. The membranes were thenexposed to primary antibody in 0.05% Tween 20 and 1%skim milk containing TN buffer for 3 h. After washing inTNbuffer containing 0.05% Tween 20 three times, the mem-branes were again incubated with blocking buffer and thenexposed to horseradish peroxidase-conjugated anti-mouseIgG (Cappel; 1:2,000) in 0.05% Tween 20/TN buffer for30 mm. Then, the membranes were washed as describedabove and soaked in ECL reagent. Peroxidase activity wasvisualized on x-ray film. Expression levels were quantifiedby the sequential dilution of samples.

Electron microscopyTheprocedures for transmission electron microscopy were

as reported previously (Takei and Endo, 1994). In brief,neurons were fixed with 2.5% glutaraldehyde in 0.1 M phos-phate buffer (pH 7.4) for 30 mm. Subsequently, neuronswere postfixed with 1% 0s04 in the same buffer for I h anddehydrated through a graded ethanol series. Epon 812 resinwas added to the samples and left to stand for 1 h. Thisprocedure was repeated twice to replace the ethanol withresin. The samples were then embedded in Epon 812 at 60°Cfor 2—3 days. Ultrathin sections were cut on a Dupont MT6000 ultramicrotome. The sections were contrasted with ura-nyl acetate and lead citrate for observation under atransmis-sion electron microscope (JEM 100 C) at an acceleratingvoltage of 80 kV.

RESULTS

Effects of BDNF on release of glutamate andGABA

Figure 1 shows the amounts of glutamate and GABAreleased from cultured neurons. There is little differ-ence in the amount of basal release of glutamate be-tween control and BDNF-treated neurons. In contrast,high-K + -evoked release of glutamate from BDNF-treated neurons was largely increased compared withthat of the control (Fig. 1A). Similar results were ob-served in the experiment of GABA release (Fig. 1B).

.1. Neurochem., Vol. 68, No. I, 1997

372 N. TAKEI ET AL.

FIG. 1. Effect of BDNF on the high K~-evokedneurotransmitterrelease in cultured cortical neurons from embryonic rats. Neu-rons were cultivated with or without BDNF (50 ng/ml) for 5 days.The amount of glutamate (A) and GABA (B) released into bufferfor 3 mm was measured by HPLC-fluorescence monitoring. Dataare mean ±SD (bars) values (n = 5). °p<0.001 versus control(CONT) high-K~by Student’s f test.

Although it was not statistically significant, BDNFtended to increase high-K + -evoked GABA release.High-K + -evoked release of both amino acids was Ca2~dependent (data not shown). The ratio of evoked/basalrelease of glutamate was 2.30 in control neurons and3.72 in BDNF-treated neurons. In GABA release, theratio was 1.6 in control neurons and 2.15 in BDNF-treated neurons. These results indicate BDNF in-creased the regulated, stimulation-evoked release butdid not affect the basal release of amino acid neuro-transmitters.

Effects of neurotrophins on levels of synapticproteins

Effects of BDNF and NT-3 on the levels of synapticproteins were investigated by western blot analysis(Fig. 2), and the levels of proteins were quantified bydensitometry (Fig. 3). Both neurotrophins increasedlevels of vesicle-associated proteins such as synapto-brevin-2 (VAMP-2), synaptophysin, synaptotagmin I,and rab3A and membrane-associated proteins such assyntaxin (HPC-l) and SNAP-25; levels of these mole-cules all increased over twofold. BDNF and NT-3 alsoincreased levels of /3-SNAP, a soluble synaptic protein,to a lesser extent. Despite the effects of BDNF andNT-3 on various exocytosis-associated proteins, not allthe synaptic proteins were affected. The levels of GAP-43, which is known as a growth cone- or synapse-associated protein, were not changed by BDNF or NT-3. Tubulin and actin levels also remained unchanged.

We confirmed that the increased levels of these pro-teins by BDNF and NT-3 were not due to the increasednumber of surviving neurons in culture by MAP-2 im-munocytochemistry. BDNF and NT-3 had no effect onthe number of total neurons in this culture system (datanot shown). In addition, there were no observabledifferences in the number or morphology of neurites

between control and neurotrophi n-treated neurons.Therefore, the observed increases in levels of theseexocytosis-associated proteins by BDNF or NT-3 rep-resented the net increases in amounts of these proteinsper neuron.

Effects of BDNF on numbers of cored and clearvesicles

The effects of BDNF on the numbers of cored andclear vesicles at the varicosities or the nerve terminalswere investigated by electron microscopy. Photomi-crographs were taken at randomly chosen varicositiesor terminals, and the numbers of vesicles were counted.Typical photomicrographs of cored vesicles and clearvesicles in terminals or varicosities are shown in Fig.4. The size of cored and clear vesicles is thought to be50—200 and ~—‘50nm in diameter, respectively (Kelly,1993). The cultured cortical neurons observed in thisstudy had cored vesicles whose size was ‘—~l00nm andclear vesicles whose size was ~-~50 nm, as shown inFig. 4A and B and C and D, respectively. The numberof dense-cored vesicles and clear vesicles was in-creased by BDNF (Fig. 5). Note that because not allthe neurons have TrkB, the effect of BDNF on thenumber of vesicles in a certain site, which was selectedrandomly, is quite different.

FIG. 2. Western blot analysis of the levels of synaptic proteinsin cultured cortical neurons from embryonic rats. Neurons werecultivated for 5 days in the absence (CONT) or presence ofBDNF (50 ng/ml) or NT-3 (50 ng/ml) and then prepared forwestern blotting.

J. Neurochem., Vol. 68, No. 1, 1997

BDNF EFFECTS ON SYNAPTIC TRANSMISSION 373

FIG. 3. Densitometric analysis of the levels of synaptic proteinsin cultured cortical neurons from embryonic rats. Neurons werecultivated for 5 days in the absence or presence of BDNF (50ng/ml; solid columns) or NT-3 (50 ng/ml; cross-hatched col-umns). Relative levels of synaptic proteins were determined bywestern blotting and analyzed by densitometry. Data are ex-pressed as fold increase over control values. Control levels arerepresented as 1.” Data are mean ±SD (bars) values (n = 4).

DISCUSSION

Many studies have shown that neurotrophic factorspromote the differentiation, maturation, and survivalof neurons both in vivo and in vitro. However, littlehas been reported concerning the long-term effects ofneurotrophins on synaptic functions. Among them,storage and release of neurotransmitters are crucialprocesses for synaptic transmission. We have shownthat intracellular contents of ACh were increased byNGF and that this increase was greater than the induc-tion of ChAT (Takei et al., 1988). Furthermore, regu-lated release of ACh was also increased by NGF incultured basal forebrain neurons (Takei et a!., 1989).These results suggested that NGF promotes the matura-tion of the neurotransmitter storage and release mecha-nisms. In this study, the effect of BDNF on the releaseof glutamate and GABA from cortical neurons wasinvestigated. Whereas only little increase was observedin basal release of both amino acid transmitters byBDNF, high-K~-evoked release of glutamate andGABA was largely increased. These results suggestthat BDNF increases the regulated release of theseneurotransmitters.

Recently, the molecular mechanisms of exocytosisof neurotransmitters were clarified, and many proteinsinvolved in exocytosis have been identified (Sudhof,1995). Therefore, to investigate the molecular basisof the up-regulation of stimulation-evoked release of

neurotransmitters, the effects of BDNF and NT-3 onthe levels of exocytosis-associated proteins were exam-ined. BDNF and NT-3 significantly increased levels ofvesicle-associated and synaptic membrane-associatedproteins, which are involved in exocytosis.

The numbers of cored and clear vesicles in nerveterminals or varicosities were also increased by BDNF.Clear vesicles are thought to contain amino acids orACh, whereas cored vesicles contain neuropeptides oramines. In cortical neurons, a majority of neurons haveglutamate or GABA as a neurotransmitter (Ottersen andStorm-Mathisen, 1984; Kaneko and Mizuno. 1994).According to the immuriocytochemical staining usinganti-glutamic acid decarboxylase and anti-glutaminaseantibodies, 30—40% of neurons are GABAergic, andalso 30—40% of neurons are glutamatergic in ourculturesystem (data not shown). From the release experiments,BDNF is suggested to act Ofl glutamatergic and probablyon GABAergic neurons in the cultured cortical neurons.Therefore, it is likely that clear vesicles, levels of whichwere increased by BDNF, may contain glutamate andGABA. BDNF markedly induced the expression of neu-ropeptide Y (Nawa et a!., 1993; Takei et a!., 1996),which is a neuropeptide colocalized with GABA in cor-tical neurons. The greater increase of the number ofcored vesicles is in good correlation with these previousreports. It is of great interest to determine whether therelease of peptide neurotransrnitters is also altered byneurotrophins.

The increases in the levels of exocytosis-associatedproteins and synaptic vesicles may, at least in part,explain up-regulation of stimulation.-evoked release ofneurotransmitters. Induction of the expression of ionchannels, which are closely associated with the trans-mitter release, by neurotrophins have also been re-ported (Mandel eta!., 1988; Toledo-Aral et al., 1995).Thus, there is a possibility that the induction of ionchannels by BDNF in this culture system may also playimportant roles in up-regulation of regulated release. Inaddition to the induction of gene expression, phosphor-ylation of synaptic proteins may also be involved inthe up-regulation. Forexample. although it was a short-term regulation, synapsin I was phosphorylated by neu-rotrophins (Jovanovic et a!., 1996).

BDNF and NT-3 had no effect on the number oftotal neurons or the number of neurites as revealed byMAP-2 immunocytochemistry. These results suggestthat under these culture conditions, i.e., high cell den-sity and serum-containing medium, neurons from em-bryos can survive and grow neurites. This is also sup-ported by the observation that the levels of tubulin andactin are unchanged by neurotrophins. Furthermore,the level of GAP-43 was not affected by BDNF orNT-3, and there was little change in the formation ofgrowth cones evoked by BDNF as revealed by scan-ning electron microscopy (data not shown). These re-sults may indicate that neurotrophins have no effecton neuritogenesis in this system. It was of interest todetermine whether the number of synapses was af-

.1. tW’lH(y hem.. Vol. 6S, No. 1, 1997

374 N. TAKEI ET AL.

FIG. 4. Electron micrographs show terminals or varicosities of cortical neurons. Neurons were cultivated in the absence (A and C) orpresence (50 ng!ml) of BDNF (B and D) for 5 days. Many cored vesicles are observed in the BONE-treated neuron (B), whereas fewvesicles appear in the control neuron (A).Arrows indicate typical cored vesicles. Many clear vesicles are observed in the BDNF-treatedneuron (D), whereas few clear vesicles appear in control neurons. Arrowheads indicate typical clear vesicles. Note that not all thevesicles are indicated by arrows and arrowheads. Bars = 1 tim.

fected by BDNF. However, this was unclear from theresults of this study because a 5-day culture periodmay be insufficient toallow synapse formation. Furtherinvestigations will be necessary for the study of synap-togenesis. It is also interesting to determine whether

BDNF affects the regulated release and the levels ofsynaptic proteins in adult brain.

In conclusion, the present results raise the possibilitythat neurotrophins up-regulate the stimulation-evokedneurotransmitter release by increasing levels of exo-

FIG. 5. Effect of BDNF on the number ofcored (A) and clear (B) vesicles in termi-nals or varicosities of cultured neurons.Cultures were maintained with or without(Cont) BONE (50 ng/ml) for 5 days. “Site”means nerve terminals and/or varicosi-ties. Each point represents the number ofvesicles. Horizontal bars represent meanvalues.

J. Neurochem., Vol. 68, No. 1, 1997

BDNF EFFECTS ON SYNAPTIC TRANSMISSION 375

cytosis-associated proteins and synaptic vesicles incortical neurons.

Acknowledgment: We thankRegeneron for the kind giftsof BDNF and NT-3. We also thank Dr. H. Murofushi (Uni-versity of Tokyo) for kindly supplying anti-MAP-2 antiseraand Dr. H. Kuramoto (Kyoto lnstitute of Technology) forhis helpful discussion.

REFERENCES

Alderson R. F., Alterman A. L., Barde Y.-A., and Lindsay R. M.(1990) Brain-derived neurotrophic factor increases survival anddifferentiated functions of rat septal cholinergic neurons in cul-ture. Neuron 5, 297—306.

Bradford M. M. (1976) A rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principleof protein—dye binding. Anal. Biochem. 72, 248—254.

Croll S. D., Wiegand S. J., Anderson K. D., Lindsay R. M., andNawa H. (1994) Regulation of neuropeptides in adult rat fore-brain by the neurotrophins BDNF and NGF. Eur. J. Neurosci.6, 1343—1353.

Hatanaka H., Tsukui H., and Nihonmatsu I. (1988) Developmentalchange in the nerve growth factor action from induction ofcholine acetyltransferase to promotion of cell survival in cul-tured basal forebrain cholinergic neurons from postnatal rats.Dcv. Brain Res. 39, 85—95.

Hefti F., Hartikka J., Eckenstein F., Gnahan H., Heumann R., andSchwab M. (1985) Nerve growth factor increases choline ace-tyltransfera.se but not survival or fiber outgrowth of culturedfetal septal cholinergic neurons. Neuroscience 14, 55—68.

Hyman C., Hofer M., Barde Y.-A., Juhasz M., Yancopoulos G.,Squinto S. P., and Lindsay R. M. (1991) BDNF is a neuro-trophic factor for dopaminergic neurons of the substantia nigra.Nature 350, 230—232.

Jovanovic J. N., Benfenati F., Siow Y. L., Sihra T. S., Sanghera J. S.,Pelech S. L., Greengard P., and Czernik A. J. (1996) Neuro-trophins stimulate phosphorylation of synapsin I by MAP kinaseand synapsin l—actin interactions. Proc. NatI. Acad. Sci. USA93, 3679—3683.

Kaneko T. and Mizuno N. (1994) Glutamate-synthesizing enzymesin GABAergic neurons of the neocortex: a double immunofluo-rescence study in the rat. Neuroscience 61, 839—849.

Kelly R. B. (1993) Storage and release of neurotransmitters. Cell72/Neuron 10 (Suppl.), 43—53.

Knusel B., Winslow J. W., Rosenthal A., Burton L. E., Seid D. P.,Nikolics K., and Flefti F. (1991) Promotion of central choliner-gic and dopaminergic neuron differentiation by brain-derivedneurotrophic factor but not neurotrophin 3. Proc. Natl. Acad.Sci. USA 88, 961—965.

Lindsay R. M., Wiegand S. J., Altar C. A., and DiStefano P. 5.(1994) Neurotrophic factors: from molecule to man. TrendsNeurosci. 17, 182—190.

Mandel G., Cooperman S. S., Maue R. A., Vicentini L. M., and

Brehm P. (1988) Selective induction of brain type II Na chan-nels by nerve growth factor. Proc. Nod. Acad. Sci. USA 85,924—928.

Mizuno K., Carnahan J., and Nawa H. (1994) Brain-derived neuro-trophic factor promotes differentiation of striatal GABAergicneurons. Dcv. Biol. 165, 243—256.

Nawa H., Bessho Y., Carnahan J., Nakanishi S., and Mizuno K.(1993) Regulation of neuropeptide expression in cultured cere-bral cortical neurons by brain-derived neurotrophic factor. J.Neurochem. 60, 772—775.

NawaH., PelleymounterM. A., andCarnahanJ. (1994) Intraventric-ular administration of BDNF increases neuropeptide expressionin newborn rat brain. J. Neurosci. 14, 3751 —3765.

Oho C., Seino S., and Takahashi M. (1995) Expression and cotnplexformation of soluble N-ethyl-maleimide-scnsitive factor atlach-ment protein (SNAP) receptors in clonal rat endocrine cells.Neurosci. Lett. 186, 208—210.

Ottersen 0. L. and Storm-Mathisen J. (1984) Glutamate- andGABA-containing neurons in the mouse and rat brain, as dem-onstrated with a new immunocytochemical technique. J. COFnO.Neurol. 229, 374—392.

Sudhof T. C. (1995) The synaptic vesicle cycle: a cascade of pro-tein—protein interactions. Nature 375, 645—653.

Takahashi M., Arimatsu Y., Fujita S.. Fujimoto Y.. Kondo S.. HamaT., and Miyamoto E. (1991) Protein kinase C and Ca/cal-modulin-dependent protein kinase II phosphorylate a novel 58-kDa protein in synaptic vesicles. Brain Res. 551, 279—292.

Takei N. and Endo Y. (1994) Ca2~ionophore-induced apoptosis on

cultured embryonic rat cortical neurons. Brain Ret. 625, 65—70.

Takei N., Tsukui H., and Hatanaka H. (1988) Nerve growth factorincreases the intracellular content of acetylcholine in culluredseptal neurons from developing rats. J. Neurochem. 51, 1118—1125.

Takei N., Tsukui H., and Hatanaka H. (1989) Intracellular storageand evoked release of acetylcholine from postnatal rat basalforebrain cholinergic neurons in culture with nerve growth fac-tor. J. Neurochem. 53, 1405—1410.

Takei N., Kondo J., Nagaike K., Ohsawa K., Kato K., and Kohsaka5. (1991) Neuronal survival factor from bovine brain is identi-cal to neuron-specific enolase. J. Neurochem. 57, 1178—1184.

Takei N., Sasaoka K., Higuchi H.. Endo Y., and Hatanaka H. ( 1996)BDNF increases the expression of neuropeptide Y mRNA andpromotes differentiation/maturation of neuropeptide Y-positivecultured cortical neurons from embryonic and postnatal rats.Mol. Brain Res. 37, 283—289.

Toledo-Aral J. J., Brehm P., Halegoua S., and Mandel G. (1995) Asingle pulse of nerve growth factor triggers long-term neuronalexcitability through sodium channel gene induction. Neuron 14,607—611.

Yoshida A., Oho C., Omori A., Kuwahara R.. Ito T.. and TakahashiM. (1992) HPC-I is associated with synaptotagmin and w-conotoxin receptor. J. Biol. Chem. 267, 24925—24928.

Widmer H. R. and Hefti F. (1994) Stimulation of GABAergic neu-ron differentiation by NT-4/5 in cultures of rat cerebral cortex.Dcv. Brain Rex. 80, 279—284.

J. Nejoochem., Vol. 68, No. I, 1997