Ž .Brain Research 832 1999 179–183

Short communication

Enhancement of activity-dependent calcium increase by neurotrophin-4 invisual cortex pyramidal neurons

Nobuo Kato a,b,), Tomoharu Tanaka a, Kenji Yamamoto a, Yoshikazu Isomura a

a Department of IntegratiÕe Brain Science, Kyoto UniÕersity Graduate School of Medicine, Kyoto 606-8501, Japanb Japan Science and Technology Corporation, Saitama 332-0012, Japan

Accepted 30 March 1999

Abstract

Ž .In pyramidal neurons from rat visual cortex slices, bath-application of NT-4 20 ngrml did not much affected the baseline calciumŽ .signal, but did enhance calcium signals elicited by injections of depolarizing currents q0.5 nA, 1 s . This enhancing effect of NT-4 was

abolished by co-applying K252a. With ryanodine injected intracellularly, the effect of NT-4 was significantly reduced, suggesting aninvolvement of intracellular calcium release in this NT-4-induced enhancement of calcium transient. q 1999 Elsevier Science B.V. Allrights reserved.

Keywords: Calcium signal; Neocortex; Calcium-induced calcium release

Ž .Neurotrophins NTs have been known to promote neu-w xronal development, differentiation, and survival 19 . In

addition, very acute effects of NTs in synaptic transmis-w xsion has recently been recognized 16,17,20,21,27 . These

synaptic effects of neurotrophins are not only rapid inw xaction, but also long-lasting 9,12,30 . Concerned with

such long-term effects of NTs, an autocrine regulation ofpostsynaptic neurotrophin release has been proposedw x1,7,8,25 . On the postsynaptic side, calcium increase is

w xknown as a prerequisite for neurotrophin release 7 . Neu-rotrophins might hence exert its autocrine actions throughmodulating intracellular calcium transients in postsynaptic

Ž .neurons. In fact, brain-derived neurotrophic factor BDNFhas been reported to increase intracellular calcium concen-tration in the dendrites and cell bodies in electricallynon-stimulated, resting hippocampal neurons in culturew x7,24 . In resting neurons, however, such postsynapticcalcium increase may be due to NT-induced enhancementof spontaneous transmitter release from presynaptic termi-

w xnals, as suggested by Canossa et al. 7 . We thus tried tostudy effects of NT-4, another ligand for TrkB, on post-synaptic calcium increases elicited by depolarization of

) Corresponding author. Department of Integrative Brain Science, Ky-oto University Graduate School of Medicine, Kyoto 606-8501, Japan.Fax: q81-75-753-4486; E-mail: f50207@sakura.kudpc.kyoto-u.ac.jp

single whole cell-clamped neurons. This method wouldallow us to investigate effects of NT-4 on calcium in-creases dependent largely on postsynaptic activity.

Ž .Young rats Wister; 10–15 days old were used. Afterprompt decapitation, the whole brain was removed and

Ž . Žincubated into a medium pH 7.4; 2–58C containing in.mM : NaCl 124, KCl 3.3, KH PO 1.3, NaHCO 26,2 4 3

CaCl 2.5, MgSO 2.0, and glucose 10. A block of the2 4Žoccipital cortex was cut and mounted on a slicer DTK-

.1000, Dosaka ME, Kyoto, Japan with agar. Slices wereŽ .cut at 200 mm in cold medium 2–58C with a razor blade.

After at least 1 h incubation at room temperature, sliceswere placed in a recording chamber attached on the stage

Ž .of an up-right microscope BHWI, Olympus with a =40Ž .water-immersion objective WPlanFl 40=UV, Olympus .

The chamber was continuously perfused at the rate ofŽ .5–10 mlrmin with medium 308C bubbled with a mixture

of O and CO . For recording, we used patch pipettes2 2Ž .resistance, 4–8 MV filled with the calcium sensitive dye

Ž .fluo-3 Dojindo, Kumamoto, Japan; 125 mM dissolvedŽ . Ž .into a solution pH 7.3 containing in mM : KCl 7, KOH

155, D-glucuronate 144, EGTA 1, Hepes 10. For fura-2ratiometry, we used an internal solution that contained 1

Ž .mM fura-2 Dojindo and no EGTA and was otherwise thesame as that for fluo-3. After making a giga-seal, thewhole-cell recording was performed under current clampŽ .Is0; Axoclamp-1D, Axon . In the meantime, the dye

0006-8993r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved.Ž .PII: S0006-8993 99 01474-2

( )N. Kato et al.rBrain Research 832 1999 179–183180

diffused into the recorded neuron from the patch pipette.Ž .Average membrane potential was 64.4"1.4 mV S.E.M.

for all the fluo-3-filled cells. Fluorescence dyes were lit byXenon light filtered at 495 nm for fluo-3 or at 360 nm and

Ž .380 nm for fura-2 Xenon lump 75 W, Ushio, Japan . Theemission fluorescence passed through a 530-nm filter forfluo-3 and 520-nm filter for fura-2. The fluo-3 fluores-

Žcence was measured with a photomultiplier OSP-10,.Olympus attached to the microscope, which was operated

by a windows-based acquisition and analysis programŽ .OSPION, Olympus . For fura-2 ratiometry, 10 measure-

Ž .ments of 380 nm-excited fluorescence intensity F and380Ž .360 nm-excited one F were done alternately at 100 Hz360

and each F value was divided by F measured 10 ms380 360

after, and then 10 F rF values thus obtained were380 360

averaged to provide one data point. Such data points wereobtained every 5 s. The value F rF was used as the380 360

index for relative calcium increase. The area from which tomeasure fluorescence intensity was a disk-shaped area of 5mm in diameter located in the proximal dendrite or soma.The fluo-3 calcium signal was digitized at 1 kHz into12-bit steps. After the patch break-in, the fluorescence dyefluo-3 started to diffuse into the cell, so that the soma andthe proximal dendrite could become visible within a fewminutes. Since patch pipettes were positively pressuredwhile approaching the recorded cell, internal solution con-taining fluo-3 spilled over the nearby tissue. This caused avisible background. However, this spillover was rapidly

Žwashed out by perfusion of standard medium 5–10.mlrmin , and consequently the background became invisi-

ble within 10 min. The background level at this stage wasmeasured and turned out to be usually less than 1% of thecalcium signal originating from the recorded neuron.

Calcium signals were evoked by injecting a depolariz-Ž .ing current q0.5 nA, 1 s through the recording pipette

Ž .somatic stimulation . Calcium signal was recorded for 5 sin total for each session. Depolarization-induced calciumtransients were recorded for 4 s from the beginning ofdepolarization. Beforehand, the baseline calcium signalwere monitored for 1 s before the depolarization. Calcium

Ž .transient frf at a given time was expressed on a relative0Ž .scale by dividing the calcium signal f by the baseline

Ž .intensity f that was calculated on the basis of the0

recording for 1 s before depolarization. Sensitivity of thephotomultiplier was adjusted for each cell, so that controlintensity could be set at the level of about 1000 over the4096 steps which covers the whole range of measurement.Measurements of the calcium signal were done first instandard medium, and then medium was changed into an

ŽNT-4-containing one 20 ngrml, recombinant human NT-.4, Alamone Labs, Israel . In control experiments, K252a

Ž . w x200 nM, Alamone Labs 3,26 was added to mediumtogether with NT-4. We also used internal solution con-

Ž .taining ryanodine 30 mM in some of the experiments.The dye bleaching by light exposure was linearly compen-sated for each measurement. The data were presented with

averages"S.E.M., and Student’s or paired t-test was usedfor statistics.

Calcium signals elicited by injection of depolarizingŽ .currents q0.5 nA, 1 s; intrasomatic stimulation were

recorded from the soma or the very proximal regions ofthe apical dendrite of layer IIrIII pyramidal neurons in therat visual cortex. The laminar origin and pyramidal mor-phology of the recorded neurons were checked by inspec-tion under microscope. The time-course of calcium signalŽ .frf was plotted for 4 s from the beginning of depolar-0

Ž .ization Fig. 1A, B . With the same 1-s depolarization, asmall, slowly-arising increase was observed in six out of

Ž .nine neurons examined Fig. 1A , whereas a more complexŽ .pattern was seen in the other three neurons Fig. 1B . In

the latter neurons, the small increase was followed by alate, sharply arising phase of increase. Whichever patternwas observed in a given neuron, that pattern was repro-ducible at the interval of 60 s or more. Similar calciumincreases were recorded from the cell body and veryproximal regions of the apical dendrites, and treated to-gether in the description below. Bath-application of NT-4Ž .20 ngrml , by itself, did not much affected the baselinecalcium signal in two neurons: frf at 1 min after NT-40

application was 100.4% and 99.1% on the basis of fluo-3fluorometry. To confirm this finding, calcium transientswere measured for 5 min after NT-4 application by usingfura-2 ratiometry, which is a more advantageous methodfor long-lasting measurements than fluo-3-based fluorome-try. In agreement, no noticeable changes in calcium signalwere detected immediately after NT-4 application or for 5

Ž .min thereafter. The calcium signal F rF measured380 360

at 5 min after NT-4 application were 103"6% of basalŽ .F rF before the application Ns4 , which can be380 360

roughly regarded as about 3% decrease in calcium signal.On the other hand, depolarization-induced calcium signalswere enhanced with NT-4, irrespective of whether neurons

Ž .exhibited a small calcium transient Fig. 1A or a two-phaseŽ .transient Fig. 1B . In neurons showing the two-phaseŽ .increase Fig. 1B , the early phase appeared to be much

Ž .less affected by NT-4 than the late phase Fig. 1B . Thiseffect of NT-4 was abolished by co-application of K252a,a broad-spectrum kinase inhibitor that blocks TrkB recep-

Ž .tors Fig. 1C .To evaluate the effects of NT-4 more quantitatively,

Ž .calcium signal frf averaged over 4 s starting from the0

beginning of depolarization was calculated. For each neu-ron, the frf values were obtained at least three times0

before NT-4 application, and also at least three times at10–30 min after the application. The average over threemeasurements before NT-4 application was compared withthat obtained after the application. The average frf was0

Ž .significantly larger after NT-4 application 1.41"0.10Ž .than before 1.17"0.04, Ns9, p-0.01 . This differ-

ence was still significant, even if the neurons which exhib-Ž .ited a small, one-phase transient e.g., Fig. 1A alone wereŽgrouped and statistically tested 1.28"0.07 after NT-4

( )N. Kato et al.rBrain Research 832 1999 179–183 181

Fig. 1. Calcium transients induced by depolarization for 1 s in layer IIrIIIŽ . Ž .pyramidal neurons from the rat visual cortex. A, B Calcium signals f

Ž .were recorded in standard medium black and NT-4-containing mediumŽ . Ž . Ž .20 ngrml; red , and expressed as ratio fr f to the baseline level f .0 0

Ž .The calcium transient consisted of a small increase alone A or took aŽ .two-phase pattern B , in which the initial small increase was followed by

a later, sharply-arising phase. In both cases, calcium increase was en-hanced by bath-application of NT-4. In B, red and black lines almostcoincide during the first 0.5 s of depolarization but not afterwards. In thisand the following Figs, the horizontal bars near the x-axis indicate the

Ž .duration of depolarization. C Calcium transients recorded in standardŽ . Žmedium black and in medium containing both NT-4 and K252a 200.nM; red .

application vs. 1.12"0.04 before the application, Ns6,.p-0.03 . When K252a was applied together with NT-4,

the average increase no longer showed a significant en-Ž .hancement 86"6% of the pre-application level; Ns6 ,

suggesting that NT-4-induced enhancement of calciumtransients was mediated by TrkB receptors.

As illustrated in Fig. 1B, the later phase of calciumtransient appeared to be affected by NT-4 to a greaterextent. To see the time-course of NT-4 effects during oneepisode of depolarization-induced calcium transient, wecalculated the mean frf separately for four consecutive0

500 ms time-windows, starting from the beginning of theŽ1-s-long depolarization. The mean frf averaged over0

.nine cells before NT-4 application were compared withthe mean frf obtained at 10–30 min after the application0Ž .Fig. 2 . This comparison, made for each of the fourtime-windows, revealed that the mean frf was signifi-0

cantly larger after NT-4 application, than before, for thesecond to forth time-windows only, but not for the first

Žwindow: 1.10"0.01 vs. 1.13"0.04 before vs. after, first. Žtime-window , 1.18"0.04 vs. 1.39"0.11 second time-

. Žwindow, p-0.03 , 1.26"0.07 vs. 1.51"0.12 third,. Žp-0.03 and 1.24"0.06 vs. 1.50"0.12 forth, p-

.0.01 . If NT-4 had augmented calcium influx via voltage-gated calcium channels alone, this effect should have beendetectable from the earliest time-window. Mechanismsdifferent from direct calcium influx are thus implicated.

A most presumable candidate of such mechanismswould be calcium-induced calcium release, which has beenwell documented in both central and peripheral neuronsw x28 . To check whether the effect of NT-4 on intracellularcalcium increases depends on calcium release, we recordedcalcium signals with patch pipettes containing the calcium

w xrelease channel blocker ryanodine 28 . Fig. 3A showscalcium transients recorded from a ryanodine-injected neu-

Ž . Ž .ron before black and after NT-4 application red . Toevaluate dependence of the NT-4 effect on ryanodine, weexpressed the mean frf after NT-4 application as percent0

of the mean frf before NT-4 application, and compared0Ž .this percentage between ryanodine-injected Ns9 and

Ž . Žuninjected neurons Ns5 for each time-window Fig..3B, increase of mean frf . The percentages were 103"0

Ž4% vs. 100"2% uninjected vs. ryanodine-injected, first. Žtime-window , 118 " 8% vs. 98 " 1% second time-

. Ž .window , 121"9% vs. 100"2% third and 121"8%Ž .vs. 104"5% forth . Except for the first time-window, the

increase of frf by NT-4 was statistically smaller in0Žryanodine-injected neurons than in uninjected neurons p

.-0.03 or 0.04; Fig. 3B . Thus, ryanodine-sensitive cal-cium stores on the postsynaptic side were modulated byNT-4.

Application of NT-4 at the concentration of 20 ngrmlfailed to increase baseline calcium concentration in the

w xpresent experiments. Unlike Canossa et al. 7 , in whichanother TrkB ligand BDNF at 100 ngrml increased cal-cium in resting neurons, the present concentration of NT-4

( )N. Kato et al.rBrain Research 832 1999 179–183182

Ž .20 ngrml was ineffective. One possibility is that ourconcentration was simply lower than needed to increasecalcium in resting neurons. Also, NT-4 may have a lowerpotency to induce calcium increase than BDNF. Alterna-tively, cultured hippocampal neurons may have differentproperties than neocortical neurons in situ. In culturedcerebellar granule cells, however, exposure to 50 ngrmlBDNF has been reported to leave the baseline calcium

w xunchanged 5 .The present results suggested that NT-4 may have

targets of its calcium-promoting action in postsynapticneurons. Such targets could be machinery for either cal-cium influx or intracellular calcium release. In the presentrecordings, NT-4 application affected the late phase ofcalcium transient but not the initial phase, and ryanodinediminished this NT-4 effect on the late phase. It is there-fore suggested that the initial and late phases of calciumtransients are largely attributable to calcium influx andcalcium release, respectively, and that NT-4 effects wereexerted on intracellular calcium release rather than oncalcium influx. Our results thus agree with Levine et al.w x18 in which BDNF, another TrkB ligand, has no effect ondepolarization-induced calcium influx.

Involvement of NTs, particularly TrkB ligands, insynaptic plasticity are widely documented in the CNSw x6,12,14,15,22,23 . Intracellular calcium increase is re-quired to induce synaptic plasticity, and this calcium in-crease is thought to originate partly from intracellular

w xcalcium release 2,11,13 . It is then suggested that TrkBligands may facilitate induction of synaptic plasticity byenhancing intracellular calcium release.

Fig. 2. Comparison of depolarization-induced calcium increases beforeŽ .and after NT-4 application. The mean of calcium signal fr f was0

obtained for each of four consecutive time-windows lasting 500 ms,starting from the initiation of the 1 s-long depolarization. The four

Ž .columns from left to right, for each of the two clusters before and after ,represent the mean fr f for the first to fourth time-windows. NT-40

application left fr f largely unchanged for the first time-window, but0

increased fr f significantly for the second to fourth time-windows.0

Error bars: S.E.M. Significance levels: U p-0.03, UU p-0.04.

Fig. 3. Dependence on ryanodine of NT-4-induced enhancement ofŽ . Ž .calcium transient. A The mean fr f before black and after NT-40

Ž . Ž . Ž .application 20 ngrml; red in a ryanodine-injected neuron 30 mM . BNT-4 effects on mean fr f in ryanodine-injected and uninjected neu-0

rons. The 2-s period, starting from the initiation of depolarization, wasdivided into four 500 ms time-windows. For each time-window, the meanfr f after NT-4 application was expressed as percent of the mean fr f0 0

Ž .before NT-4 application increase of mean fr f , and was averaged over0Ž .ryanodine-injected Ryanodine, Ns5 and uninjected neurons separately

Ž .No Ryanodine, Ns9 . Difference in the percentage between the ryan-odine-injected and uninjected groups was significant for the second toforth time-windows, but not for the first window. Error bars: S.E.M.Significance levels: U p-0.03, UU p-0.04.

An autocrine regulation is proposed as a mechanism forNT secretion, in which secreted NTs activate further secre-tion of the same NTs from postsynaptic cellsw x1,4,7,8,10,25,29 . Secretion of NTs is generally thought to

w xdepend on intracellular calcium release 7 . Therefore, ifactivity-dependent calcium release is enhanced in the pres-ence of NT-4, as suggested in the present report, secretedNT-4 might facilitate further secretion of NT-4 by enhanc-ing intracellular calcium release. Secreted NT-4 would alsoup-regulate synaptic transmission, which would in turncontribute to reinforcing activity-dependent intracellular

( )N. Kato et al.rBrain Research 832 1999 179–183 183

calcium release. By this way, autocrine secretion of NT-4may be cooperatively linked with maintenance of up-regu-lated synaptic efficiency.

Acknowledgements

This work was supported by a PRESTO project of JST,and by grants from the Ministry of Education, Science andCulture of Japan to N.K. We thank Dr. M. Sokabe andX.F. Zhou for comments.

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