brain-derived neurotrophic factor induces long-lasting ca2+-activated k+ currents in rat visual...

Brain-derived neurotrophic factor induces long-lastingCa2+-activated K+ currents in rat visual cortex neurons

Yoshito Mizoguchi,1,2 Akira Monji2 and Junichi Nabekura1

1Department of Cellular and Systems Physiology, and2Department of Neuropsychiatry, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan

Keywords: Ca2+ imaging, pyramidal cell, PLC-g, TrkB receptor, tyrosine kinase

Abstract

Brain-derived neurotrophic factor (BDNF) increases postsynaptic intracellular Ca2+ and modulates synaptic transmission in

various types of neurons. Ca2+-activated K+ currents, opened mainly by intracellular Ca2+ elevation, contribute tohyperpolarization following action potentials and modulate synaptic transmission. We asked whether BDNF induces Ca2+-

activated K+ currents by postsynaptic elevation of intracellular Ca2+ in acutely dissociated visual cortex neurons of rats. Currents

were analysed using the nystatin-perforated patch clamp technique and imaging of intracellular Ca2+ mobilization with fura-2. At aholding potential of ±50 mV, BDNF application (20 ng/mL) for 1±2 min induced an outward current (IBDNF-OUT; 80.0 6 29.0 pA)

lasting for more than 90 min without attenuation in every neuron tested. K252a (200 nM), an inhibitor of Trk receptor tyrosine

kinase, and U73122 (3 mM), a speci®c phospholipase C (PLC)-g inhibitor, suppressed IBDNF-OUT completely. IBDNF-OUT was both

charybdotoxin- (600 nM) and apamin- (300 nM) sensitive, suggesting that this current was carried by Ca2+-activated K+ channels.BAPTA-AM (150 mM) gradually suppressed IBDNF-OUT. Fura-2 imaging revealed that a brief application of BDNF elicited a long-

lasting elevation of intracellular Ca2+. These results show that BDNF induces long-lasting Ca2+-activated K+ currents by sustained

intracellular Ca2+ elevation in rat visual cortex neurons. While BDNF, likely acting through the Trk B receptor, was necessary forthe induction of long-lasting Ca2+-activated K+ currents via intracellular Ca2+ elevation, BDNF was not necessary for the

maintenance of this current.

Introduction

Neurotrophins play important roles in neuronal differentiation,

neurite outgrowth and survival as well as in the maintenance of

matured neurons (Thoenen, 1991). These effects generally occur over

the course of hours to days. Recent studies indicate that neurotrophins

can rapidly in¯uence synaptic transmission within minutes after the

application (Thoenen, 1995; Li et al., 1998). Imaging of intracellular

Ca2+ revealed that BDNF, a neurotrophin that binds with high af®nity

to Trk B tyrosine kinase receptor, increases intracellular Ca2+ within

minutes in various types of neurons and other cells (Berninger et al.,

1993; Montcouquiol et al., 1997; Kleiman et al., 2000; Matsumoto

et al., 2001; Numakawa et al., 2001). In rat hippocampus, BDNF

acutely inhibits GABAA-mediated responses caused by postsynaptic

elevation of intracellular Ca2+ through the activation of Trk B

receptors (Tanaka et al., 1997). BDNF also elevates intracellular Ca2+

and induces long-term potentiation (LTP) of excitatory postsynaptic

potentials (EPSPs) in the hippocampus (Kang & Schuman, 2000) and

visual cortex (Akaneya et al., 1997; Jian et al., 2001). LTP of

inhibitory postsynaptic potentials (IPSPs) also requires a postsynaptic

intracellular Ca2+ increase for induction (Komatsu, 1996; Komatsu &

Yoshimura, 2000).

Ca2+-activated K+ currents are found in most types of cells (Blatz

& Magleby, 1987) and normally contribute to membrane hyperpolar-

ization following action potentials in neurons. They are thought to be

involved in setting the resting membrane potential and ®ring

frequencies (Sah, 1996), as well as modulating synaptic activity

and transmitter release (Robitaille et al., 1993). In rat hippocampus,

Ca2+-activated K+ channels modulate LTP of EPSPs (Sah & Bekkers,

1996; Behnisch & Reymann, 1998). The Ca2+-activated K+ channels,

opened mainly by intracellular Ca2+ elevation, are classi®ed into

three families based on their single channel conductance (Vergara

et al., 1998). The large conductance (BK) Ca2+-activated K+

channels are both voltage- and intracellular Ca2+-sensitive. The

small (SK) and intermediate (IK) conductance Ca2+-activated K+

channels are opened solely by intracellular Ca2+ and are more

sensitive to Ca2+ than BK channels (Latorre et al., 1989). Thus, it is

possible that BDNF induces Ca2+-activated K+ currents due to

intracellular Ca2+ elevation in rat visual cortex neurons. To address

this possibility, nystatin-perforated patch clamp recordings and

imaging of intracellular Ca2+ mobility were performed in acutely

dissociated rat visual cortex neurons. We found that BDNF induced

long-lasting Ca2+-activated K+ currents by sustained elevation of

intracellular Ca2+, most likely through the activation of Trk B

tyrosine kinase receptors.

Materials and methods

All experimental protocols conformed to the Guiding Principles for

the Care and Use of Animals approved by the Council of the

Physiological Society of Japan. All efforts were made to minimize

both the number of animals used.

Correspondence: Dr J. Nabekura, as above.E-mail: [email protected]

Received 22 March 2002, revised 25 July 2002, accepted 30 July 2002

doi:10.1046/j.1460-9568.2002.02198.x

European Journal of Neuroscience, Vol. 16, pp. 1417±1424, 2002 ã Federation of European Neuroscience Societies

Preparation

Twelve- to fourteen-day-old Wistar rats were decapitated under

pentobarbital anaesthesia. Brains were quickly removed and trans-

versely sliced at a thickness of 380 mM (DM IRB, Leica, Germany).

The slices were kept in the incubation medium saturated with 95%

O2/5% CO2 at room temperature (22±25 °C) for at least 1 h. To

dissociate cortical neurons, the slices were transferred into a 35-mm

culture dish (Primaria 3801, Becton Dickinson, NJ, USA) and layer V

of the visual cortex was identi®ed under a binocular microscope

(SMZ-1, Nikon, Tokyo, Japan). A ®re-polished glass pipette was

touched lightly onto the surface of layer V region and was vibrated

horizontally at 3±5 Hz for 3 min using an apparatus developed in our

laboratory (Kakazu et al., 2000). Slices were removed from the dish

and mechanically dissociated cortical pyramidal-like neurons adhered

to the bottom of the dish within 20 min.

Electrical measurements

Electrical measurements were performed using the nystatin-

perforated patch recording method (Nabekura et al., 1993; Omura

et al., 1999). All recordings were performed using voltage clamp at a

holding potential of ±50 mV, using a patch clamp ampli®er (EPC-7,

List Electronics, Germany). This holding potential was chosen as it

was close to the typical resting membrane potential of these cells yet

suf®ciently apart from the K+ equilibrium potential to enable us to

record K+ currents with good signal-noise ratio. Patch pipettes were

made from borosilicate capillary glass tubes (1.5 mm outside

diameter, 9 mm insude diameter; G1.5, Narishige, Tokyo, Japan) in

two stages on a vertical pipette puller (PB-7,Narishige). Nystatin-

perforated patch recordings were employed on neurons visualized

with phase contrast equipment on an inverted microscope

(DIAPHOT TMD300,Nikon,Tokyo,Japan). Current and voltage

were monitored on an oscilloscope (VC-6725, Hitachi, Tokyo,

Japan) as well as a pen recorder (Recti±Horiz 8K, Nippondenki

San-ei, Tokyo, Japan). Membrane currents were ®ltered at 1 kHz

(E-3201 A Decade Filter, NF Electronic Instruments, Tokyo, Japan)

and data were digitized at 4 kHz. To measure the reversal potential of

the outward current induced by BDNF application, ramp voltage

steps from ±50 to ±140 mV of 1500 ms duration were applied using a

function generator (MacLab, AD Instuments, Australia; Kakazu et al.,

1999). Data were also simultaneously collected using computer

software (SCOPE V3.6, AD Instruments, Australia). All experiments

were performed at room temperature (22±25°C).

Solutions

The ionic composition of the internal (patch pipette) solution was

(in mM); 40 methanesulfonic acid potassium salt, 110 KCl, 10

HEPES. The pH of internal solution was adjusted to 7.2 with Tris-

OH. Nystatin was dissolved in acidi®ed methanol at 10 mg/mL. The

stock solution was diluted with internal pipette solution just before

use to a ®nal concentration of 100±200 mg/mL. The resistance of

the recording electrode ®lled with internal solution was 4±6 MW. The

ionic composition of the incubation medium was (in mM); 124

NaCl, 5 KCl, 1.2 KH2PO4, 24 NaHCO3, 2.4 CaCl2, 1.3 MgSO4 and 10

glucose. The pH of the incubation medium was adjusted to 7.4

with 95% O2/5% CO2. The ionic composition of the external

standard solution was (in mM); 150 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2,

10 glucose and 10 HEPES. The pH of the external standard solution

was adjusted to 7.4 with Tris-OH. Ca2+-free external solution was

obtained by simply removing CaCl2 from the external standard

solution.

Drugs

Drug solutions were applied, using the `Y-tube' method which

allowed us to exchange the external solution within 20 ms (Nabekura

et al., 1996). Drugs used included charybdotoxin (Peptide Institute,

Osaka, Japan), tetraethylammonium (TEA; Tokyo Kasei, Tokyo,

Japan), apamin (Peptide Institute, Osaka, Japan), and 4-aminopyr-

idine(4-AP; Tokyo Kasei). BAPTA-AM (Calbiochem, Los Angeles,

CA, USA) was initially dissolved in dimethylsulfoxide (DMSO) and

then diluted in the external solution.

Human recombinant BDNF (PeproTech House, London, England)

was dissolved (100 mg/mL) in phosphate buffer solution containing

0.1% BSA and stored below ±20 °C. Before the experiment, the stock

solution was diluted with external solution to the ®nal concentration

(20 ng/mL). K252a (Calbiochem, La Jolla, CA, USA) and U73122

(Calbiochem) were ®rst dissolved in DMSO. The ®nal concentration

of DMSO < 0.1% did not affect neuronal responses observed in the

present study.

Ca2+ imaging

Intracellular Ca2+ levels in response to BDNF application were

monitered using fura-2 AM (acetoxymethyl ester; Grynkiewicz et al.,

1985) in acutely dissociated visual cortex neurons of rats. Neurons

were loaded with 5 mM Fura-2 AM (Calbiochem), a membrane-

permeable Ca2+ indicator dye, for 30 min at 37 °C and were washed

three times with the external standard solution before measurement

using digital video imaging ¯uorescence microscopy (403 magni®-

cation, with ECLIPSE E600FN, Nikon). During measurement,

external standard solution was constantly perfused (1 mL/min).

Images were captured at excitation wavelengths of 340 and 380 nm

and were stored every 2±5 s. The ratio of ¯uorescence at the two

exciting wavelengths (F) was calculated for each pixel within a cell

boundary (METAFLOUR software, Universal Imaging corporation,

Downingtown, PA, USA). The ratio (F/F0) of ¯uorescence intensity

was estimated from the intensity of ¯uorescence before compared to

after BDNF application. BDNF (20 ng/mL) and Ca2+-free external

solution were applied in the perfusate (6 mL/min). All Ca2+ imaging

experiments were carried out at room temperature (24±25 °C).

Results

BDNF induces a long-lasting outward current in rat visualcortex neurons

We tested whether BDNF induces current responses in rat visual

cortex neurons using nystatin-perforated patch clamp recordings,

which allow intracellular substances such as Ca2+ and proteins to

remain intact. All recordings were performed under the voltage-

clamp mode, at a holding potential of ±50 mV. BDNF (20 ng/mL)

induced a long- lasting outward current (IBDNF-OUT) with an increase

of membrane conductance in every neuron tested (n = 12; Figs 1 and

3B). An increase of input resistance was also con®rmed by use of

hyperpolarizing voltage pulses at intervals of 60 s (from ±50 mV to

±60 mV, 300 ms in duration; data not shown). In every neuron, the

time to onset was constantly 1±2 min after the start of BDNF

application. The amplitude of IBDNF-OUT gradually increased and

reached a steady state level (80 6 29.0 pA, means 6 SE; n = 12)

within 5±8 min after the onset of the current. IBDNF-OUT persisted for

more than 90 min without attenuation until the end of the recording.

Interestingly, once IBDNF-OUT occurred, it gradually increased in

amplitude to a steady state level and persisted without attenuation,

regardless of the presence (n = 3; Fig. 1A) or the absence (n = 9;

Fig. 1B) of BDNF in the extracellular solution. These results suggest

1418 Y. Mizoguchi et al.

ã 2002 Federation of European Neuroscience Societies, European Journal of Neuroscience, 16, 1417±1424

that BDNF induces a long-lasting outward current in rat visual cortex

neurons, but is not necessary for its maintenance.

Possible involvement of Trk B receptor tyrosine kinase activityin the induction and maintanence of IBDNF-OUT

BDNF speci®cally binds to Trk B, a neurotrophin receptor, contain-

ing a catalytic domain of tyrosine kinase (Thoenen, 1995). We next

examined the involvement of Trk B receptor tyrosine kinase activity

in the induction and the maintenance of IBDNF-OUT.

In the presence of K252a (200 nM), a membrane-permeable

inhibitor of Trk receptor tyrosine kinase, BDNF (20 ng/mL) failed

to induce a current (n = 3; Fig. 2A). In addition, K252a (200 nM)

completely abolished the steady state current of IBDNF-OUT (Fig. 2B).

This inhibition persisted more than 10 min after the end of the K252a

application. K252a (200 nM) did not change the base-line current

(Fig. 2A). This result suggests that K252a-sensitive pathway, such as

Trk B receptor tyrosine kinase activity, is involved in the induction as

well as the maintanence of IBDNF-OUT.

Ionic mechanisms of IBDNF-OUT

To determine the charge carrier of IBDNF-OUT, hyperpolarizing

voltage ramp commands were applied before and during IBDNF-OUT

(Fig. 3A). The reversal potential of IBDNF-OUT was ±83.8 6 2.5 mV

(mean 6 SE; n = 10), close to the theoretical K+ equilibrium

potential (±85.6 mV) calculated from Nernst equation under our

experimental conditions ([K+]out, 5 mM; [K+]in, 150 mM) (Fig. 3B).

Thus, BDNF induces IBDNF-OUT by opening K+ channels.

To characterize the K+ channels opened by BDNF, we examined

the effects of various K+ channel blockers on IBDNF-OUT in the steady

state (Fig. 4A and B). Charybdotoxin (600 nM), a BK and IK

FIG. 2. Possible involvement of Trk B receptor tyrosine kinase activity in the induction and maintenance of IBDNF-OUT. (A) In the presence of K252a(200 nM; open bar), a membrane-permeable inhibitor of Trk receptor tyrosine kinase, BDNF (20 ng/mL) failed to induce a current. (B) IBDNF-OUT wascompletely suppressed by a 2-min application of 200 nM K252a. K252a application started 10 min after the onset of IBDNF-OUT. Dashed line indicates thebase-line current level.

FIG. 1. BDNF induces outward currents in rat visual cortex neurons. (A) 20 ng/mL BDNF (closed bar) induced an outward current (IBDNF-OUT) at a holdingpotential of ±50 mV. BDNF was applied for 24 min. The amplitude of IBDNF-OUT gradually increased to a steady state level and persisted without attenuation.Note that IBDNF-OUT was stably maintained with continual application of BDNF. (B) The application of 20 ng/mL BDNF for 3 min also induces an outwardcurrent. After washout of BDNF, IBDNF-OUT gradually increased and reached a steady state level, which persisted without attenuation of the current for morethan 90 min (see also Fig. 3A). Dashed line indicates the base-line current level.

Ca2+-activated K+ currents induced by BDNF 1419


FIG. 4. Involvement of Ca2+-activated K+ channels in IBDNF-OUT. (A) IBDNF-OUT were sensitive to both 600 nM charybdotoxin (Ch-Tx) and 10 mM TEA.4-Aminopyridine (4-AP; 3 mM) did not affect IBDNF-OUT. (B) IBDNF-OUT were partially blocked by 300 nM apamin. An additional application of 10 mM TEAto apamin completely suppressed IBDNF-OUT to the base-line current level. Dashed line indicates the base-line current level before the onset of IBDNF-OUT. Thetraces in A and B were obtained from different neurons. (C) The current±voltage relationships for IBDNF-OUT under 300 nM apamin application showedvoltage dependence.

FIG. 3. Ionic mechanisms of IBDNF-OUT. (A) To examine the reversal potential of IBDNF-OUT, voltage ramp commands of ±90 mV were applied before (a) andduring (b) IBDNF-OUT. The holding potential was ±50 mV (B) The current±voltage relationships for voltage ramps with (b) and without (a) BDNF. Thereversal potential of IBDNF-OUT (EIBDNF-OUT) is indicated as the membrane potential (Vm) at which the two current responses intersected each other.EIBDNF-OUT was close to the theoretical reversal potential of K+ (EK+ ±85.6 mV) calculated from external(5 mM) and internal K+ concentrations (150 mM).



channels blocker, suppressed IBDNF-OUT 34.3 6 4.4% (mean 6 SE;

n = 6), while 10 mM TEA, a BK and IK channels blocker, caused

38.5 6 3.0% (n = 5) and 300 nM apamin, a selective SK channel

blocker, caused 60.6 6 1.2% (n = 3) suppression, respectively. On

the other hand, 4-AP (3 mM), a nonselective voltage gated K+

channel blocker, did not affect IBDNF-OUT (n = 3), but adding TEA

(10 mM) to apamin (300 nM) completely suppressed IBDNF-OUT to the

base-line current level (n = 3; Fig. 4B). Every K+ channel blocker

used did not affect the base-line current (data not shown). To examine

the voltage sensitivity of IBDNF-OUT, ramp voltage steps from ±120 to

+20 mV of 2000 ms duration were applied in the presence of

apamin(300 nM). The current±voltage relationships for apamin-

insensitive IBDNF-OUT showed voltage dependence (n = 3; Fig. 4C).

These results suggest that BDNF activates BK and SK, Ca2+-

activated K+ channels sensitive to charybdotoxin and apamin.

Sustained intracellular Ca2+ elevation for the maintenance ofIBDNF-OUT

To address the relationship between intracellular Ca2+ mobilization

and IBDNF-OUT, we examined the effect of 1,2-bis(2-aminophenoxy)-

ethane-N,N,N¢,N¢-tetraacetic acid, AM ester (BAPTA-AM), a

membrane-permeable Ca2+ chelator. BAPTA-AM (150 mM) slowly

suppressed IBDNF-OUT from the steady state level to the base-line level

(n = 4; Fig. 5). This suggested that sustained intracellular Ca2+

elevation was necessary for the maintenance of IBDNF-OUT.

Induction of a long-lasting intracellular Ca2+ elevation byBDNF

Next, we tested BDNF-induced intracellular Ca2+ mobility using

fura-2. As shown in Fig. 6, BDNF (20 ng/mL) increased intracellular

Ca2+ in visual cortex neurons. The time between the start of BDNF

application and the onset of Ca2+ elevation (1±3 min) was similar to

that of IBDNF-OUT (Fig. 1). The increase in intracellular Ca2+ concen-

tration was sustained for > 50 min even after the washout of BDNF

(n = 7). This suggests that BDNF induces a long-lasting intracellular

Ca2+ elevation, which supports the electrophysiological observations

that BDNF induces a long-lasting Ca2+-activated K+ current.

To examine the involvement of extracellular Ca2+ in the mainten-

ance of long-lasting intracellular Ca2+ elevation, we applied Ca2+-free

standard extracellular solution after intracellular Ca2+ reached the

maximal elevation. Removal of extracellular Ca2+ did not affect the

intracellular Ca2+ elevated by BDNF (Fig. 6). This result suggests

that extracellular Ca2+ is not important for the maintenance of the

long-lasting intracellular Ca2+ elevation.

The involvement of PLC-g phosphorylation in the maintenanceof IBDNF-OUT

BDNF binds to the Trk B receptor and activates many intracellular

signalling pathways including PLC-g (Patapoutian & Reichardt,

2001), which generates inositol triphosphate and mobilizes intra-

cellular Ca2+ from endoplasmic reticulum in rat cortex (Widmer et al.,

1993). We next examined the involvement of intracellular Ca2+

mobilization via PLC-g phosphorylation in the maintenance of IBDNF-

OUT.

U73122 (3 mM), a membrane-permeable speci®c PLC-g inhibitor

(Yule & Williams, 1992), slowly suppressed IBDNF-OUT from the

steady state level to the base-line level (n = 3; Fig. 7) and

intracellular Ca2+ elevated by BDNF (n = 3, data not shown). This

result suggests that PLC-g phosphorylation is involved in the

maintenance of IBDNF-OUT and intracellular Ca2+ elevation by BDNF.

Discussion

The present experiments demonstrated that BDNF induces a

sustained elevation of intracellular Ca2+ and long-lasting Ca2+-

activated K+ currents in rat visual cortex neurons due to the activation

of K252a-sensitive receptor tyrosine kinases, probably Trk B. Our

results also show that BDNF is required for the induction of the long-

lasting Ca2+-activated K+ currents as well as sustained intracellular

Ca2+ elevation, but is not required for their maintenance.

BDNF induces long-lasting Ca2+-activated K+ currents in ratvisual cortex neurons

In visual cortex, long-term potentiation (LTP) has been proposed as a

synaptic basis for experience-dependent changes in the structure and

the function of neural circuits (Tsumoto, 1992). In developing visual

cortex, neuronal activity is required for the maintenance of LTP at

inhibitory synapses (Komatsu & Yoshimura, 2000) and LTP requires

postsynaptic Ca2+ accumulation originating from internal Ca2+ stores

rather than from an extracellular source (Komatsu, 1996). BDNF

plays an important role in formation and activity-dependent modi-

®cation of neural circuits of visual cortex during postnatal develop-

ment (Thoenen, 1995; Bonhoeffer, 1996). More rapid actions of

BDNF on neuronal function include that acute enhancement of

synaptic transmission and roles in synaptic plasticity, including LTP

in rat visual cortex (Akaneya et al., 1997). In hippocampus, Ca2+-

activated K+ channels modulate LTP (Sah & Bekkers, 1996; Behnisch

& Reymann, 1998). We showed that BDNF induced a long-lasting

Ca2+-activated K+ currents in postsynaptic neurons at visual cortex.

FIG. 5. Effect of Ca2+ chelator on IBDNF-OUT. The application of 150 mM BAPTA-AM for 10 min (dotted bar) gradually suppressed IBDNF-OUT to the base-linecurrent level (dashed line). Thus, a sustained elevation of intracellular Ca2+ is necessary for the maintenance of IBDNF-OUT.



The Ca2+-activated K+ currents causes prolonged after-hyperpolar-

izations (Sah, 1996; Vergara et al., 1998). Thus, the Ca2+-activated

K+ currents induced by BDNF could reduce calcium in¯ux through

NMDA receptors via prolonged afterhyperpolarizations or by shunt-

ing EPSPs and modulate LTP in visual cortex.

We demonstrated that BDNF induced Ca2+-activated K+ currents

caused by intracellular Ca2+ elevation likely through the activation of

Trk B receptor tyrosine kinase and PLC-g phosphorylation. In rat

hippocampus, BDNF acutely reduces the amplitude of IPSCs by

postsynaptic Ca2+ elevation through the stimulation of PLC-gphosphorylation (Tanaka et al., 1997). PLC-g, activated by Trk B,

generates inositol triphosphate which mobilizes intracellular Ca2+

from endoplasmic reticulum in rat cortex (Widmer et al., 1993).

NT-3 and nerve growth factor (NGF), but not BDNF, enhance

TEA-sensitive BK currents elicited by voltage steps from 0 to

100 mV in rat cortical culture neurons (Holm et al., 1997). Regarding

the intracellular mechanisms of the NT-3 effect, they showed that

NT-3 stimulates PLC-g through the activation of Trk C receptor,

resulting in dephosphorylating the BK channels or another key

protein without an increase of intracellular Ca2+ level. We assumed

that BDNF elevates intracellular Ca2+ concentration and sequentially

induces Ca2+-activated K+ currents. Involvement of intracellular Ca2+

elevation in the activation of Ca2+-activated K+ channels by

neurotrophin remains to be studied.

BDNF is not required for the maintenance of long-lastingCa2+-activated K+ currents

A brief application of BDNF has been shown to induce long-lasting

potentiation of EPSPs in rat visual cortex in vivo (Jiang et al., 2001).

This is similar to the present observation that a brief BDNF

application induces a long-lasting intracellular Ca2+ elevation and

Ca2+-activated K+ currents. Here, we report that BDNF is required for

induction, but not maintenance. In the cortex, a brief release of

neurotrophin is associated with synaptic activity (Altar & DiStefano,

1998; Kohara et al., 2001).

Brief application of NGF, one of the neurotrophins, also induces a

long-lasting action (Toledo-Aral et al., 1995). A 1-min application of

NGF induces voltage-activated sodium currents lasting more than

24 h in PC12 cells. A brief application of NGF rapidly phosphor-

ylates PLC-g and elevates intracellular Ca2+ levels through Trk A

receptor tyrosine kinase activation, in which the autophosphorylation

of Trk A receptor and phosphorylation of PLC-g are sustained for up

to 30 min and 2 h, respectively (Choi et al., 2001). Because

neurotrophins have similar signalling features (Barbacid, 1995), the

long-lasting Ca2+ elevation resulting in the activation of Ca2+-

activated K+ currents, might be attributable to the sustained activation

of intracellular signalling cascades such as autophosphorylation of

tyrosine kinase and phosphorylation of PLC-g. Indeed, both K252a

FIG. 7. The involvement of PLC-g in the maintenance of IBDNF-OUT. IBDNF-OUT was slowly suppressed by a 3-min application of 3 mM U73122 (diagonallystriped bar), a membrane-permeable PLC-g inhibitor. U73122 application started 18 min after the onset of IBDNF-OUT.

FIG. 6. Induction of a long-lasting intracellular Ca2+ elevation by BDNF. The changes of intracellular Ca2+ concentration after BDNF application weremeasured by Fura-2 imaging. The traces shown are the representative of seven reproducible experiments. The application of 20 ng/mL BDNF induced a rapidrise in intracellular Ca2+ concentration. The application of Ca2+-free external standard solution for 15min (open bars) did not have any effect on the elevatedintracellular Ca2+ levels. Thus, the maintenance of long-lasting intracellular Ca2+ elevation did not require external Ca2+.



and U73122 suppressed the steady state of IBDNF-OUT in the absence

of BDNF (Figs 2B and 7). The removal of extracellular Ca2+ did not

affect the sustained increase in intracellular Ca2+ evoked by BDNF

(Fig. 6). Sustained activations of tyrosine kinase and PLC-g by a brief

application of NGF has been demonstrated (Choi et al., 2001). Thus,

it seems that a brief application of BDNF also caused a sustained

activation of PLC-g in visual cortex neurons, leading to a sustained

release of Ca2+ from internal stores. We presume that the sustained

constant level of elevated Ca2+ results from some equilibrium

between the constant Ca2+ release and the various Ca2+ extrusion

mechanisms. However, we cannot rule out the possibility that BDNF

also impairs the ability of various Ca2+ buffering mechanisms to

return intracellular Ca2+ to resting levels.

In conclusion, the results presented here demonstrate that BDNF

induces a sustained elevation of intracellular Ca2+ and long-lasting

Ca2+-activated K+ currents in rat visual cortex neurons due to the

activation of K252a-sensitive receptor tyrosine kinases, probably

Trk B. Our results also show that BDNF is required for the induction

of the long-lasting Ca2+-activated K+ currents as well as sustained

intracellular Ca2+ elevation, but is not required for their maintenance.

These rapid and sustained actions of BDNF might play an important

role in neuronal plasticity and long-lasting changes of neuronal

excitability in developing visual cortical neurons.

Acknowledgements

We would like to give our appreciation to Dr Rita J. Balice-Gordon atUniversity of Pennsylvania for critical reading of the manuscript. This work issupported by Grants-in-Aid for Scienti®c Research 13210108 on AdvancedBrain Research and 13035036 on Integrated Brain Research (to J.N.) from theMinistry of Education, Culture, Sports and Science and Technology, Japan.

Abbreviations

BDNF, brain-derived neurotrophic factor; LTP, long-term potentiation;DMSO, dimethylsulfoxide; TEA, tetraethylammonium; 4-AP, 4-aminopyr-idine; Ch-Tx, charybdotoxin; PLC, phospholipase C.

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