Ž .Brain Research 815 1999 140–149

Interactive report

BDNF-dependent enhancement of exocytosis in cultured cortical neuronsrequires translation but not transcription 1

John Bradley, Olaf Sporns )

The Neurosciences Institute, 10640 John Jay Hopkins DriÕe, San Diego, CA 92121, USA

Accepted 12 October 1998

Abstract

Ž .Neurotrophic factors, such as brain-derived neurotrophic factor BDNF , are involved in acute modulation of synaptic plasticity.Different modes of action of BDNF have been described with time courses ranging from seconds to hours, but the sequence of cellularprocesses responsible for BDNF-dependent modulation of synaptic plasticity is unknown. We have used optical imaging of the styryl dye,FM1-43, which selectively labels synaptic vesicles, to investigate potential presynaptic effects of BDNF. Addition of BDNF to culturedcortical neurons for 3 h produced a significant enhancement of exocytosis upon modest depolarization. BDNF had no effect on exocytosiseither immediately or after incubation for 30 min. BDNF-dependent enhancement of exocytosis was blocked by the tyrosine kinaseinhibitor, K252a, but not by K252b, consistent with signalling via the TrkB receptor. Having demonstrated that the BDNF-dependentenhancement of synaptic vesicle release was present only after 1 h, we investigated whether de novo gene transcription andror proteinsynthesis were involved. Addition of the inhibitors of RNA synthesis, actinomycin D, or 5,6-dichloro-1-b-D-ribofuranosyl benzimidazoleŽ .DRB , did not affect the enhancement of exocytosis produced by BDNF. However, the effect of BDNF was blocked by the inhibitors oftranslation, cycloheximide or anisomycin. Our results indicate a rapid BDNF-dependent enhancement of neurotransmitter release thatrequires translation but not transcription. q 1999 Elsevier Science B.V. All rights reserved.

Keywords: Brain-derived neurotrophic factor; Exocytosis; Synaptic vesicle; FM1-43; Neurotransmitter; Synaptic plasticity

1. Introduction

Neurotrophic factors, such as brain-derived neu-Ž .rotrophic factor BDNF , are not only involved in the

w xsurvival and differentiation of neurons 1,2 , but they alsow xmodulate synaptic plasticity 3,4 . Application of BDNF to

w x w xhippocampal 5,6 and cortical slices 7,8 , to the dendatew xgyrus of the anaesthetized rat 9 , and to cultured neurons

w x10,11 produces potentiation of synaptic transmission.w xBDNF also facilitates the induction 12 and enhances the

w x Ž .expression 7 of long term potentiation LTP . Thesefindings, together with studies showing that neurotrophinscan be expressed and released in an activity-dependent

w x w xmanner 13,14 , possibly from synaptic sites 15 , indicatethat BDNF may function as an extracellular messengerwhich may modulate synaptic function in response to

w xneuronal activity 16,17 .

) Tel.: q1 q 619-626-2000; Fax: q1-619-626-2199; E-mail:sporns@nsi.edu

1 Published on the World Wide Web on 9 November 1998.

The mechanisms by which BDNF increases the efficacyof synaptic transmission are still largely unknown. BDNFhas been shown to increase the frequency but not theamplitude of miniature excitatory postsynaptic currentsŽ . w xmEPSCs 8,10,18 , reduce the effect of paired–pulse

w xfacilitation 5,19 , increase the variance of the amplitudesw xof evoked EPSCs 20 , and attenuate synaptic fatigue

w x12,21 . These observations, which are believed to reflectthe efficiency of synaptic vesicle release during exocytosis,provide indirect evidence that BDNF affects presynaptic

w xfunction. Although Levine and colleagues 11 showed thatinhibition of postsynaptic TrkB receptors blocked BDNF-dependent enhancement of synaptic transmission, a recent

w xstudy 22 demonstrated that elimination of functionalpresynaptic, but not postsynaptic, TrkB receptors was suf-ficient and necessary to prevent enhancement of synaptic

w xtransmission by BDNF. Wang and Poo 23 showed thatŽ .postsynaptic overexpression of neurotrophin-4 NT-4 ,

Ž .produced modulation of acetylcholine ACh receptors,presumably by an autocrine action on postsynaptic TrkBreceptors. In the same study the authors reported an effectof NT-4 on presynaptic function, but unlike the modulationof ACh receptors, this effect did not require sustained

0006-8993r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved.Ž .PII: S0006-8993 98 01112-3

( )J. Bradley, O. SpornsrBrain Research 815 1999 140–149 141

w xrelease of NT-4 23 . It appears therefore that BDNF mayhave both pre- and postsynaptic effects, but the sequenceof events responsible for the potentiation of synaptic trans-mission and LTP, and the time course over which theybecome apparent, are still not well understood. In thepresent study we investigated specifically the effect ofexogenous application of BDNF on a presynaptic process,vesicular neurotransmitter release, and the time courseover which a BDNF-dependent effect may occur.

Ž ŽThe fluorescent styryl dye FM1-43 N- 3-triethylam-. Ž Ž . .moniumpropyl -4- 4- dibutylamino styryl pyridinium di-

. w xbromide selectively labels synaptic vesicles 24 and hasbeen used in a number of studies to investigate presynapticfunction in various experimental paradigms known to po-

w xtentiate synaptic transmission 25,26 . We used FM1-43 toinvestigate the effect of BDNF on stimulated exocytosis ofsynaptic vesicles. We present evidence that addition ofBDNF has no immediate effect on exocytosis but enhancesvesicular neurotransmitter release within 1–3 h, possiblyby a TrkB-dependent mechanism. Analysis of individualsynaptic sites reveals that BDNF exerts a potentiatingeffect on a large number of synapses causing a significantshift in the population response. BDNF-induced potentia-

Ž .tion of exocytosis requires translation protein synthesisbut appears to be independent of de novo gene transcrip-tion.

2. Materials and methods

2.1. Cell culture

All experiments were carried out in accordance withprotocols approved by the Institutional Animal Care andUse Committee of the Neurosciences Institute and withfederal and state policies. Primary cultures of cortical

Žneurons were prepared from E19 embryonic rats Wistar. w xKyoto, Harlan using methods previously described 27 .

Briefly, 4–6 cerebral hemispheres were dissected, connec-tive tissue was removed, and the pooled tissue was incu-

Žbated in Hanks’ balanced salt solution HBSS,2q 2q . Ž .Ca rMg -free; Gibco containing trypsin 0.1% at 378C

for 10 min. The tissue was washed 3 times withCa2qrMg2q-free HBSS before the cells were dissociatedwith a fire-polished Pasteur pipette. After allowing thedebris to settle the supernatant was removed. Calcium andmagnesium were returned to the buffer and cells were

Ž .centrifuged 2 min at 1500 g . The supernatant was dis-carded and the pellet was resuspended in Neurobasal

Ž . Ž .medium Gibco containing B27 supplement Gibco , glu-Ž . Ž .tamine 500 mM and glutamate 25 mM . No serum,

antibiotics or antimitotic agents were present during cul-ture. Cells were subsequently plated at a density of 300

2 Žcellsrmm onto coverslips coated with poly L-lysine 1.mgrml , and were maintained at 378C in a humidified

environment of 95% airr5% CO . After 4 days in vitro2

Ž .DIV , one-half of the medium was removed and replacedwith glutamate-free medium.

2.2. FM1-43 labelling and drug treatment

Following 9–12 days of culture, activity-dependentdestaining was monitored using the styryl dye FM1-43Ž . w xMolecular Probes as described previously 27 . Cells

Žwere mounted in a laminar superfusion chamber volume.of 400 ml on a temperature-controlled microscope stage

Ž . Ž .358C and continuously superfused 1 mlrmin with salineŽcontaining, mM; NaCl 119, KCl 2.5, MgCl 2, CaCl 2,2 2

. ŽHEPES 25, and glucose 30, pH 7.4 . FM1-43 dissolved in.water and 0.2 mm filtered was loaded at a concentration

of 10 mM in saline containing 90 mM Kq. 1 min of dyeapplication was followed by 5–10 min of washing withsaline to reduce background staining. Exocytosis was stim-ulated by the perfusion of saline containing Kq at various

Ž .concentrations 30 mM–90 mM . To prevent recurrentneuronal activity upon stimulation with Kq, all fluores-cence measurements were made in the presence of theglutamate receptor antagonists 6-cyano-7-nitro-quinoxa-

Ž .line-2,3-dione CNQX, 10 mM; RBI, Natick, MA andŽD,L-2-amino-5-phosphonovaleric acid AP-5, 50 mM;

.Sigma . All pharmacological reagents, including K252aŽand K252b, anisomycin, actinomycin D all Calbiochem,

. ŽSan Diego, CA , and cycloheximide Sigma, St. Louis,.MO , were added to the culture medium 30 min before the

beginning of an experiment; human recombinant BDNFŽ .Alexis Biochemicals, San Diego, CA was added to the

Ž .culture medium from a stock solution 100 mgrml andcells were incubated for specific time periods.

2.3. Fluorescence imaging

All fluorescence measurements were taken with a LeicaŽDM-IRBE inverted epifluorescence microscope Leica,

.Deerfield, IL , equipped with a 50 W mercury light sourceusing the 63 = r0.7 NA Fluotar objective. Excitation wasthrough a 450–490 nm band-pass filter, and emission was

Ždetected through a 520 nm long-pass filter. Images grey.scale, 8-bit resolution were acquired with a cooled CCD

Žcamera Optronics DEI-470T, Optronics Engineering, Go-. Žleta, CA at a rate of one image per 2.5 s 2 s integration

.timerimage and stored digitally for later data analysis. Nopre-processing was applied to the images and care wastaken that all relevant parts of the image remained in the

Žlinear range this was accomplished by ensuring that mini-mal fluorescence values of pixel intensities were well

.above 0 and maximal values were well below 255 . Tominimize potential phototoxic effects all cells wererecorded within a few seconds of the onset of fluorescentillumination.

2.4. Data analysis

Time courses of destaining sequences were analysedŽusing MetaMorph software Universal Imaging Corpora-

( )J. Bradley, O. SpornsrBrain Research 815 1999 140–149142

.tion, West Chester, PA as follows. When necessary, stacksof images were aligned manually to correct for minorlateral movements of the image during a run; runs whichshowed any movements of the focal plane were rejected.

ŽMultiple regions of interest 1 mm = 1 mm squares, 8 = 8.pixels were centred on bright fluorescent punctae, and for

each region an equally-sized second region was definedcovering a nearby area of background. The total fluores-

Ž .cence intensities grey level of all regions of interest were

recorded for all images within the run and the data wasŽthen analysed using Microsoft Excel Microsoft Corpora-

.tion, Redmond, WA . For each region of interest, thecorresponding background region was subtracted. We esti-mated the vesicular release due to depolarization as fol-lows. We computed a baseline fluorescence by averagingthe fluorescence intensity of regions of interest within 4

Ž .video frames 2.5 s apart, total times10 s that wererecorded just before the onset of depolarization. Then, we

q Ž . Ž . qFig. 1. K -evoked exocytosis in populations of synapses of cortical neurons. A Stimulation of destaining by 30 mM and 90 mM shaded histogram Kpulses applied for 30 s. The histograms represent the combined distribution of destains from multiple independent experiments expressed as the % destainŽ .see Methods ; for each experiment between 70 and 250 FM1-43-positive sites from all parts of the image were evaluated. The number in the upper left is

Ž . qthe percentage of non-destainable sites first histogram bin for 30 mM K ; nsnumber of experiments, ss total number of recorded synapses, and theŽ . Ž . Ž . Ž . Ž .dashed line corresponds to a baseline distribution ns5, ss802 . B Cumulative plots of % destain distributions for 30 mM m , 55 mM n , 65 mM r ,

Ž . q Ž .and 90 mM l K pulses, all applied for 30 s. The dashed line no symbol is a cumulative plot of a baseline distribution. Each treatment was applied to atleast 4 different cell preparations. The vertical dashed line indicates the 30% destain value; the percentage of responses greater than 30% was used to

Ž . qquantify population responses. C Plot of the percentage of responses greater than 30% for different concentrations of K ; standard error of the mean isq Ž .shown. A submaximal concentration of K 30 mM was used throughout the study in order to evaluate any potentiation produced by BDNF.

( )J. Bradley, O. SpornsrBrain Research 815 1999 140–149 143

computed a ‘post-destain’ fluorescence by averaging thecorresponding fluorescence intensities within 4 video

Ž .frames, starting 10 s 4 frames after the onset of depolar-Žization. The difference between these two values ex-

.pressed as ‘percent destain’ served as our measure ofvesicular release.

3. Results

3.1. Responses of synaptic populations to K q depolariza-tion

In order to identify baseline experimental conditionsthat would allow synaptic potentiation to occur we initiallyobtained population responses across a range of Kq con-centrations. As described previously for hippocampal neu-

w xrons 27,28 , cortical neurons showed numerous fluores-Žcent punctae along their neurites following incubation 1

. qmin with FM1-43 in the presence of 90 mm K andŽ .subsequent wash-out of excess dye 5–10 min . The cap-

ture of an image series without stimulation of the cellsproduced a baseline distribution which reflects a decreasein fluorescence due to non-specific fluctuations in bright-ness or lateral movements of individual fluorescent spotsrather than actual destaining responses due to exocytosisŽ .Fig. 1, dashed line . When a depolarizing stimulus wasapplied, consisting of a 30 s pulse of Kq at 30 mm, 55mm, 65 mm or 90 mm, destaining responses occurred at

Ž .many of the previously labelled punctae Fig. 1 . While weobserved significant heterogeneity of responses within agiven population of synapses for all concentrations of Kq

Ž .Fig. 1A, Fig. 2 , individual experimental runs yieldedreproducible levels of destaining for populations of synap-

q Ž .Fig. 2. Enhancement of K -evoked exocytosis by BDNF in populations of synapses of cortical neurons. FM1-43 staining of neurites either untreated A orŽ . Ž .following a 3 h incubation with BDNF 20 ngrml B ; all images have the same scale and the scale bar is 5 mm. The images were captured immediately

Ž . Ž . q Ž . Ž . qbefore i and 20 s after ii a 30 s pulse of K . White arrows mark the same locations before i and after ii the K pulse. Sample recordings ofq Ž . Ž . Ždestaining responses during K -dependent depolarization from 12 randomly chosen sites indicated by white arrows of untreated cells C or BDNF 20

. Ž . Ž .ngrml -treated cells D are shown. Traces represent total fluorescence within an 8 = 8 pixel region 1mm x 1mm centered on a given location, withq Ž .background from a region of the same size subtracted and the data expressed as the percentage of the baseline; arrows indicate onset of K pulse. E

Ž . Ž . q Ž .Averages of the 12 individual responses for untreated and BDNF-treated cells shown in C and D ; arrow marks the onset of the K pulse. FŽ . Ž .Cumulative plot of percentage destain for the response of the population of synapses for untreated cells m, ss76 and BDNF-treated cells s, ss107 .

Ž .The distribution for BDNF-treated cells is significantly different from that of control cells P-0.01, Kolmogorov–Smirnov test, bin widths2% .

( )J. Bradley, O. SpornsrBrain Research 815 1999 140–149144

tic sites. Cumulative histograms revealed that the overallextent of destaining for the synaptic population increased

q Ž .with increasing concentrations of K Fig. 1 . To quantifyresponses of populations of synapses across experimentalconditions we used entire cumulative population curves aswell as the percentage of synapses which showed a

Ždestaining response greater than 30% indicated by the.dashed vertical line in Fig. 1B . We found that stimulation

with 30 mm Kq produced a relatively weak populationresponse while showing a significant increase in the num-ber of synapses with greater than 30% destain relative to

Žthe baseline distribution Fig. 1C; P-0.01; ns4, all Pvalues were obtained using an unpaired Student’s t-test

.unless otherwise stated . The submaximal concentration of30 mM Kq was used throughout the study in order toevaluate any potentiation produced by BDNF on stimu-lated exocytosis.

3.2. BDNF enhances K q-eÕoked exocytosis

In hippocampal slices, maximum potentiation of synap-tic transmission was observed 1 h after application of

Fig. 3. Effects of BDNF on Kq-evoked exocytosis revealed by analysis of averaged population responses. Cumulative plots of percentage destainŽ . Ž . Ž .distributions A and relative intensity distributions B obtained from cortical neurons which were either untreated m; ns4, ss473 or were incubatedŽ . Ž . qwith BDNF 20 ngrml for 3 h s; ns7; ss918 . Initial intensity values represent the fluorescence averaged over 16 s before application of a K pulse;

Ž .intensity values were scaled for each coverslip of cells. C The averaged temporal profile of destaining for synapses showing a percentage destain greaterŽ . Ž . Ž .than 30% for untreated cells fine line and BDNF-treated cells thick line . D Histogram of the percentage destains greater than 30% showing the effect

Ž . Ž . Žof tyrosine kinase inhibitors. The inhibitors K252a 200 nM and K252b 200 nM were added to the culture medium 30 min before addition of BDNF 20. qngrml ; cells were incubated with BDNF for 3 h, loaded with FM1-43, washed and then stimulated with a 30 s pulse of 30 mM K . The dashed horizontal

line in this and the following figures indicates the percentage of synapses showing destain responses greater than 30% to a 30 s pulse of 30 mM Kq

Ž . Ž .indicated as ‘‘K30’’ . All treatments were carried out on at least 3 different cell preparations except K252b, ns2 .

( )J. Bradley, O. SpornsrBrain Research 815 1999 140–149 145

w xBDNF 5 . We therefore investigated the effect of BDNFŽ . q20 ngrml on K -evoked exocytosis following a 3 hincubation. Fig. 2 compares the Kq-evoked responses of 2populations of cortical neurons, one of which was treatedwith BDNF for 3 h while the other remained untreated.Fig. 2A,B show representative examples of FM1-43 la-

Ž .belled neurites of untreated cells Fig. 2A and cells treatedŽ . Ž .with BDNF for 3 h Fig. 2B , immediately before i and

Ž . q20 s after ii the onset of a 30 s pulse of 30 mM K .Arrows indicate 12 randomly selected synaptic sites forwhich the recording traces of destaining responses are

Ž . Žshown in Fig. 2C untreated cells and Fig. 2D BDNF-.treated cells . Each trace represents the total fluorescence

within a 1 mm = 1 mm region centered on a given loca-Ž .tion after subtraction of a background region and is

expressed as a percentage of the baseline. Averages of the12 individual responses for untreated and BDNF-treatedcells are shown in Fig. 2E and cumulative plots of percent-age destains for the whole population of synapses ofuntreated and BDNF-treated cells are shown in Fig. 2F.The distribution for BDNF treated cells is significantly

Ždifferent from that of control cells P-0.01, Kol-.mogorov–Smirnov test, bin widths2% .

BDNF-dependent enhancement of exocytosis was con-sistently observed with different preparations of cells. A 3h incubation of BDNF caused a rightward shift of the

Žcumulative plot of percentage destains Fig. 3A, P-0.01;.Kolmogorov–Smirnov test, bin widths2% , and signifi-

cantly increased the percentage of destains greater thanŽ .30% P-0.01; ns7 . We also examined the effect of

BDNF on the initial labelling intensity of FM1-43-positivesites. Initial intensity of an individual background-sub-tracted region was calculated as the fluorescence averagedover 16 s before application of a Kq pulse. No overtdifference in absolute labelling intensities was observedbetween any experimental conditions. To obtain relativevalues for intensity we scaled initial intensity values be-

Ž . Ž .tween 0 minimum intensity and 100 maximum intensityfor each experimental run. Cumulative plots of relativeintensity values for all synaptic sites showed no significant

Ždifference between untreated and BDNF-treated cells Fig..3B . These data indicate that treatment of cells for 3 h with

BDNF increases the extent of destaining responses ofsynapses in a population, but the population distribution ofinitial labelling intensity remains unchanged.

In order to evaluate the effect of BDNF on the temporalprofile of destaining responses we compared averagedresponses, showing destains greater than 30%, for un-

Ž . Ž . Žtreated ns43 and BDNF-treated cells ns118 Fig..3C . No significant difference in the temporal profile of

destaining was observed following a 3 h incubation withBDNF.

There is evidence that physiological effects of BDNFare mediated by receptor tyrosine kinases, specifically

w xTrkB 1 . It has been shown that TrkB receptors arew xexpressed in cultured cortical neurons 29,30 . We there-

fore investigated the possible involvement of the TrkBreceptor in the enhancement of exocytosis produced by a 3

Ž .h incubation with BDNF 20 ngrml using 2 structurallyw xsimilar tyrosine kinase inhibitors, K252a and K252b 31 .

As shown in Fig. 3D, a 30 min preincubation of K252aŽ .200 nM completely blocked the BDNF-induced enhance-

Ž .ment of exocytosis. In contrast, K252b 200 nM , a com-Žpound which does not inhibit tyrosine kinases including

.TrkB at the concentration used, did not block the BDNF-Ž .dependent increase in exocytosis P-0.01; ns4 . This

data indicates that a tyrosine kinase-mediated signallingŽ .pathway possibly involving TrkB may be responsible for

the observed enhancement of exocytosis.

3.3. BDNF enhances exocytosis in a time-dependent man-ner

A number of studies have demonstrated that BDNFproduces changes in synaptic efficacy which become ap-

w xparent over a wide temporal range, from minutes 10 tow xdays 23,32 . We therefore investigated the time course of

BDNF-dependent enhancement of exocytosis to gain in-sight into the possible cellular mechanisms responsible forthe effect. We found that the enhancement of exocytosis

Ž .by BDNF is time-dependent. BDNF 20 ngrml did notproduce an immediate stimulation of exocytosis and no

Ženhancement was apparent after a 30 min incubation Fig..4 . However, an increase in the destaining response was

Ž .evident after 1 h P-0.08; ns4 which was highly

Fig. 4. Time-dependence of BDNF enhancement of Kq-evoked exocyto-sis. Histogram of the percentage destains greater than 30%. Superfusion

Ž q . Žof normal saline buffer no K stimulation containing BDNF 20. Ž .ngrml for 1 min solid bar produced a destaining distribution which

was not significantly different from measurements made with normalŽ . Žsaline buffer in the absence of BDNF indicated as ‘‘Basal’’ . BDNF 20

. Žngrml was added to the culture medium for the indicated time shaded.bars before the cortical neurons were loaded with FM1-43, washed and

q Ž .then stimulated with a 30 s pulse of 30 mM K indicated as ‘‘K30’’ .All experiments were carried out on at least 3 different cell preparations.

( )J. Bradley, O. SpornsrBrain Research 815 1999 140–149146

Ž .significant after 3 h P-0.02; ns9 . While almost allexperiments were carried out with BDNF present through-out the incubation period, we also tested whether an initialexposure to BDNF was sufficient to elicit potentiation ofsynaptic responses. If BDNF was removed after 30 min byreplacing the culture medium with medium lacking BDNF

Žbut containing the tyrosine kinase inhibitor K252a 200.nM , to block further signal transduction by receptor-bound

Ž .BDNF, a potentiation P-0.06; ns3 was still observed3 h after BDNF was initially added.

3.4. BDNF-dependent enhancement of exocytosis requirestranslation but not transcription

BDNF-dependent enhancement of exocytosis only be-comes apparent after at least 1 h and is still observed whenBDNF is removed after 30 min. Since this temporal profileresembled the time course of BDNF-dependent synaptic

w xpotentiation in the hippocampal slice 5 , a process shownw xto depend on protein synthesis 33 , we investigated

whether translation andror transcription are required forthe potentiation of presynaptic transmitter release. In thepresence of two structurally different inhibitors of transla-

Ž . Ž .tion, cycloheximide 40 mM and anisomycin 40 mM , noBDNF-dependent enhancement of exocytosis occurred atany time point. Note that BDNF does not produce signifi-cant increases in destaining responses when compared tountreated controls, both with and without addition of therespective translation blocker. We also note that cyclohex-imide alone appears to produce a slight decrease in exocy-tosis, an effect that is not observed with anisomycin. In thepresence of the structurally dissimilar inhibitors of tran-

Ž . Ž .scription, actinomycin D 25 mM , and DRB 100 mM ,BDNF produced an enhancement of exocytosis, with thenumber of sites destaining by more than 30% being signifi-

Ž .cantly different from the untreated cells P-0.05; ns3 .

4. Discussion

The aim of the present study was to investigate theeffect of BDNF on the exocytosis of presynaptic vesiclesfrom individually resolved synapses in populations of cor-tical neurons. Using optical imaging of neurons stainedwith the styryl dye, FM1-43, we found that exocytosisevoked by Kq-dependent depolarization was enhanced byBDNF. BDNF had no immediate effect on exocytosis,instead the effect became evident only after a period of1–3 h. We also showed evidence suggesting that theenhancement produced by BDNF may be dependent uponTrkB receptor activation and involved subsequent proteinsynthesis, but was independent of de novo gene transcrip-tion.

BDNF has been shown to enhance synaptic transmis-w x w xsion 5 and LTP 12 , but the sequence of cellular pro-

cesses responsible for the effect remains unclear and both

pre- and postsynaptic changes have been describedw x11,20,23 . Since most previous studies have used electro-physiological recording techniques, presynaptic effects ofBDNF have been inferred from postsynaptic responsesoften involving highly interconnected populations of neu-rons. In the present study, therefore, we use the styryl dyeFM1-43, an indicator of vesicular neurotransmitter releasew x24 , to monitor BDNF-dependent effects. This approachhas several advantages. First, FM1-43 fluorescence is con-fined to synaptic vesicles within the presynaptic terminalw x34 and measurements of their release potentially providean unambiguous indicator of a presynaptic BDNF effect.Second, responses of individual synaptic sites can be quan-titatively measured. Third, responses can be efficientlycollected from large populations. Previous reports usingoptical detection of FM1-43 described heterogeneity of

w xsynaptic responses 27,35 , and for this reason we evalu-ated the effect of BDNF on populations of synapses ofcortical neurons. We found that repeats of individual ex-perimental runs under identical conditions produced con-sistent levels of synaptic responses across a population.Therefore all data analysis presented in this paper wascarried out on the observed population response of typi-cally hundreds of synapses per experimental run.

Since previous reports indicated a BDNF-dependentw xpotentiation of synaptic responses 10,11 , we first deter-

q Ž .mined a K concentration 30mM , in the absence ofBDNF, that produced a submaximal stimulation of exocy-

Ž .tosis within our assay Fig. 1 . When treating corticalŽ .neurons with BDNF 20 ngrml for 3 h we found an

Žincreased number of strong destaining responses exocyto-. Ž .sis upon depolarization of the cells Fig. 2 . Comparing

large numbers of single responses obtained from individualpairs of experiments, we found a consistent increase in thepopulation response after BDNF treatment. We attributethis shift in the population response to a BDNF-dependentenhancement of exocytosis.

We examined in detail cumulative curves of destainingresponses and initial intensities for populations of synapses

Ž .across multiple sets of experiments Fig. 3 . Destainingresponses, on average, showed a parallel rightward shift

Ž .after treatment with BDNF Fig. 3A similar to the effectq Ž .produced by increasing the K concentration Fig. 1B .

However, no significant difference was observed in thedistributions of initial intensities for untreated and BDNF-

Ž .treated cells Fig. 3B . Thus, the population destainingresponse appears shifted while overall intensities for initiallabelling remained unchanged. This is consistent with adistributed effect of BDNF expressed at most, if not all,synaptic sites. On the other hand, an effect involving onlya specific subpopulation of synapses, or one resulting inthe formation of an entirely new set of sites does notappear to be evident. The latter point receives additionalsupport from the fact that no obvious structural changes ofthe cells were observed within a 1 to 3 h time frame. TheBDNF-dependent enhancement of FM1-43 destaining may

( )J. Bradley, O. SpornsrBrain Research 815 1999 140–149 147

be accounted for by an increase in the size of the re-leasable pool of synaptic vesicles at existing sites. Thishypothesis is supported by the finding that BDNF reduces

w xsynaptic fatigue 12 , which also indicates an increase inthe size of the readily-releasable pool of synaptic vesicles.Since BDNF does not increase the amplitude of sponta-

w xneous mEPSCs 8,10 , it is unlikely that there is a changein the quantum of each release event. However, additionalexperiments are needed to identify the precise mechanismby which BDNF enhances exocytosis.

The technique used in the present study enabled us toevaluate and quantify an effect of BDNF on presynapticfunction either immediately, or at multiple time pointsfollowing the application of BDNF. We initially investi-gated whether BDNF produces direct stimulation of exocy-tosis. BDNF has been reported to stimulate directly the

Ž .immediate release within minutes of ACh from synapto-w xsomes derived from septal cholinergic neurons 36 and the

w xrelease of dopamine from mesencephalic neurons 37 .However, we did not find any direct effect of BDNF on

Ž .exocytosis within our culture system Fig. 4 . This sup-w xports the conclusion of Takei and colleagues 32 , who

showed that BDNF does not directly cause exocytosis butincreases the amount of glutamate released upon Kq-de-pendent depolarization. Second, we determined the timecourse of the BDNF-dependent effect. In contrast to theeffects of BDNF revealed by whole-cell patch clamp

w xrecording 10,18 , which were evident within minutes, wedid not observe an effect of BDNF until at least 1 h after

Ž .BDNF addition Fig. 4 . This may suggest that the en-hancement of exocytosis produced by BDNF does notresult from a rapid presynaptic second messenger cascade,e.g. involving the phosphorylation of synapsin by MAP

w xkinase 38 , and that more complex signalling is requiredŽ .see below .

We have presented evidence that the BDNF-dependentenhancement of exocytosis can be blocked by inhibitors of

Žprotein synthesis but not by inhibitors of transcription Fig..5 . The requirement of newly synthesized proteins is con-

sistent with the delayed onset of BDNF-induced enhance-Ž .ment Fig. 3 . These findings are in agreement with those

w xof Kang and Schuman 5 who showed a delay in the onsetof BDNF-dependent synaptic enhancement in hippocampalslices which could also be blocked by inhibitors of protein

w xsynthesis 33 . It is possible that BDNF activates TrkBreceptors located on the soma stimulating the synthesis ofproteins that are subsequently transported within the ax-onal compartment to synaptic terminals. Although BDNFhas been demonstrated to elevate the levels of proteins

w xassociated with exocytosis 32 , these measurements weremade after 5 days of culture in the presence of BDNF.Whether the process of protein synthesis and delivery fromthe soma to axon terminals could be completed within 1 h

w xremains to be determined. Kang and Schuman 33 alsodemonstrated, using specific lesions within the hippocam-pal slice preparation, that the protein synthesis required for

Fig. 5. Requirement of translation, but not transcription, for BDNF-de-Ž .pendent enhancement of exocytosis. BDNF 20 ngrml was added to the

culture medium 3 h prior to stimulating the cells with 30 mM Kq for 30Ž . Ž . Žs shaded bars . Cycloheximide ‘‘cyclo’’; 40 mM , anisomycin ‘‘aniso’’;

. Ž . Ž .40 mM , actinomycin D ‘‘actino’’; 25 mM and DRB 100 mM were alladded to the culture medium 30 min prior to the addition of BDNF. Allexperiments were carried out on at least 3 different cell preparations.

BDNF-dependent enhancement of synaptic transmissionoccurred within the dendrites of CA1 pyramidal neurons.A number of studies have also reported protein synthesisin the dendritic compartment, a phenomenon termed local

w x Ž .protein synthesis 39,40 , and neurotrophin-3 NT-3 hasbeen shown to stimulate the relocation of mRNA to den-

w xdrites of cultured cortical neurons 41 . If, under the condi-tions of the present study, BDNF stimulated dendriticprotein synthesis, then it is interesting to speculate howenhancement of synaptic vesicle release could result fromnewly synthesized proteins located postsynaptically. Onepossible mechanism may involve the postsynaptic stimula-tion of protein synthesis by BDNF such that newly synthe-sised proteins may enhance, or stimulate the release of, aretrograde signal to potentiate vesicular release from thepresynaptic terminal. One possible candidate, a diffusiblecompound capable of modulating neurotransmitter releasew x27 , is nitric oxide.

The data of the present study unambiguously show apresynaptic effect of BDNF, the enhancement of vesicularneurotransmitter release upon Kq-dependent depolariza-tion. This provides further support for the hypothesis thatBDNF may act as diffusible extracellular messenger capa-ble of modulating synaptic function. Since the enhance-ment of vesicular release requires protein synthesis, furtherstudies will be needed to establish which proteins are

( )J. Bradley, O. SpornsrBrain Research 815 1999 140–149148

involved and whether they are located in pre- androrpostsynaptic compartments.

Acknowledgements

This study was supported by Neurosciences ResearchFoundation.

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