Reduction in Voltage Gated K Channel_J Neurosci

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*Department of Anesthesiology and Perioperative Medicine, The University of Texas MD Anderson Cancer Center, Houston, Texas,

USA

Program in Neuroscience, The University of Texas Graduate School of Biomedical Sciences, Houston, Texas, USA

Peripheral neuropathy is one of the most common compli-

cations of diabetes. One of the most prominent features of

diabetic neuropathy is the development of pain that typically

involves the extremities, occurring as an exaggerated

response to either a painful stimulus (hyperalgesia) or a

mild and normally non-painful stimulus (allodynia) (Brown

and Asbury 1984; Clark and Lee 1995). The precise cellular

mechanisms of hyperalgesia and allodynia in diabetic

neuropathic pain remain poorly understood. Increased excit-

ability of primary sensory neurons plays a critical role in

painful diabetic neuropathy (Hong et al. 2004; Jagodic et al.

2007). It has been shown that voltage-gated Na+ channel

currents are significantly increased in both small- and large-

sized dorsal root ganglion (DRG) neurons in diabetic rats

(Hong et al. 2004; Hong and Wiley 2006). There is also

a significant increase in low- and high-voltage-gated

Ca2+channel currents in DRG neurons in diabetic neuropathy

(Hall et al. 1995; Jagodic et al. 2007). However, changes in

other ion channels involved in the increased excitability of

DRG neurons in diabetic neuropathic pain are not fully

known.

Received May 6, 2010; revised manuscript received May 26, 2010;

accepted June 9, 2010.

Address correspondence and reprint requests to Hui-Lin Pan, MD,

PhD, Department of Anesthesiology and Perioperative Medicine, Unit

110, The University of Texas MD Anderson Cancer Center, 1515 Hol-

combe Blvd., Houston, TX 77030, USA.

E-mail: [email protected]

Abbreviations used: 4-AP, 4-aminopyridine; BDNF, brain-derived

neurotrophic factor; DAP, 3,4-diaminopyridine; DRG, dorsal root

ganglion; IB4, isolectin B4; NGF, nerve growth factor; PBS, phosphate-

buffered saline; Kv channels, voltage-gated K+ channels; STZ,

streptozotocin; TEA, tetraethylammonium.

Abstract

Abnormal hyperexcitability of primary sensory neurons plays

an important role in neuropathic pain. Voltage-gated potas-

sium (Kv) channels regulate neuronal excitability by affecting

the resting membrane potential and influencing the repolari-

zation and frequency of the action potential. In this study, we

determined changes in Kv channels in dorsal root ganglion

(DRG) neurons in a rat model of diabetic neuropathic pain.

The densities of total Kv, A-type (IA) and sustained delayed

(IK) currents were markedly reduced in medium- and large-,

but not in small-, diameter DRG neurons in diabetic rats.

Quantitative RT-PCR analysis revealed that the mRNA levelsof IA subunits, including Kv1.4, Kv3.4, Kv4.2, and Kv4.3, in the

DRG were reduced 50% in diabetic rats compared with

those in control rats. However, there were no significant dif-

ferences in the mRNA levels of IK subunits (Kv1.1, Kv1.2,

Kv2.1, and Kv2.2) in the DRG between the two groups.

Incubation with brain-derived neurotrophic factor (BDNF)

caused a large reduction in Kv currents, especially IA currents,

in medium and large DRG neurons from control rats. Fur-

thermore, the reductions in Kv currents and mRNA levels of IA

subunits in diabetic rats were normalized by pre-treatment

with anti-BDNF antibody or K252a, a TrkB tyrosine kinase

inhibitor. In addition, the number of medium and large DRG

neurons with BDNF immunoreactivity was greater in diabetic

than control rats. Collectively, our findings suggest that diabe-

tes primarily reduces Kv channel activity in medium and large

DRG neurons. Increased BDNF activity in these neurons likely

contributes to thereduction in Kv channel function through TrkBreceptor stimulation in painful diabetic neuropathy.

Keywords: diabetic neuropathy, dorsal root ganglion, ion

channels, neuropathic pain, neurotophic factors, voltage-

gated potassium channels.

J. Neurochem. (2010) 114, 14601475.

JOURNAL OF NEUROCHEMISTRY | 2010 | 114 | 14601475 doi: 10.1111/j.1471-4159.2010.06863.x

1460 Journal Compilation 2010 International Society for Neurochemistry, J. Neurochem. (2010) 114, 14601475

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Voltage-gated K+ (Kv) channels are important for the

regulation of the resting membrane potential, the duration

and frequency of the action potential, and the release of

neurotransmitters in neurons (Kim et al. 2005; Catacuzzeno

et al. 2008). The native Kv currents in primary sensoryneurons include two major types based on their inactivation

kinetics and sensitivities to tetraethylammonium (TEA) and

3,4-diaminopyridine (DAP) or 4-aminopyridine (4-AP):

slowly inactivating delayed currents (IK) and rapidly

inactivating transient A-type currents (IA) (Everill et al.

1998; Liu and Simon 2003; Vydyanathan et al. 2005). In the

isolectin B4 (IB4)-positive DRG neurons, the IA is particu-

larly important in the control of the spike onset, the threshold

of the action potential firing, and the firing frequency

(Vydyanathan et al. 2005). The IK is also involved in

determining the threshold of the action potential firing, the

repolarization and after-hyperpolarization phase, and the

resting potential in primary sensory neurons (Safronov et al.

1996; Catacuzzeno et al. 2008). It has been shown that

traumatic nerve injury reduces the mRNA levels of the

Kv1.1, Kv1.2, Kv1.4, Kv2.2, and Kv4.2 subunits in DRG

neurons (Kim et al. 2002) and both the IA and IK in DRG

neurons (Everill and Kocsis 1999; Abdulla and Smith 2001;

Yang et al. 2004). Nevertheless, little is known about the

changes in Kv channel activity in DRG neurons in diabetic

neuropathic pain.

Brain-derived neurotrophic factor (BDNF) is normally

present in some small- and medium-sized DRG neurons

(Zhou and Rush 1996; Thompson et al. 1999). The expres-

sion level of BDNF is increased in small-sized DRG neuronsin response to peripheral inflammation (Karchewski et al.

2002; Obata et al. 2003a). Increased BDNF expression also

occurs in axotomized medium and large DRG neurons

(Tonra et al. 1998; Obata et al. 2003b) and in the DRG in

diabetic rats (Fernyhough et al. 1995). It has been shown that

treatment with BDNF, not nerve growth factor (NGF), can

reduce the mRNA levels of IA subunits in DRG neurons

(Park et al. 2003). However, it is not clear whether BDNF

plays a role in reducing Kv channel function in DRG neurons

in diabetic neuropathy. In this study, we (i) examined the

changes in Kv currents in different sized DRG neurons in a

rat model of painful diabetic neuropathy and (ii) determined

the role of BDNF in reducing Kv channel activity in DRG

neurons in diabetic neuropathic pain.

Methods

Animal model of diabetic neuropathic pain

Male SpragueDawley rats (9 weeks old, Harlan SpragueDawley,

Indianapolis, IN, USA) were used. All experiments were approved

by the Animal Care and Use Committee of the University of Texas

M. D. Anderson Cancer Center and conformed to the guidelines of

the National Institutes of Healths Guide for the Care and Use of

Laboratory Animals. All efforts were made to minimize both the

suffering and number of animals used. Diabetes was induced by a

single intraperitoneal (i.p.) injection of 60 mg/kg streptozotocin

(STZ; Sigma, St. Louis, MO, USA) freshly dissolved in 0.9% sterile

saline (Chen and Pan 2002). Age-matched vehicle-injected rats were

used as the controls. Previous studies have demonstrated that after

STZ injection, most rats display reproducible mechanical allodynia

and hyperalgesia within 3 weeks, lasting for at least 7 weeks

(Courteix et al. 1993; Chen and Pan 2002; Khan et al. 2002). This

model of neuropathic pain mimics the symptoms of neuropathy in

diabetic patients, with alterations in pain sensitivity and poor

responses to l opioid administered systemically or intrathecally

(Courteix et al. 1993; Malcangio and Tomlinson 1998; Zurek et al.

2001; Chen and Pan 2002; Khan et al. 2002). Diabetes was

confirmed in the STZ-injected rats by measurement of the blood

glucose concentration. The glucose level in the blood from the tail

vein was assayed using ACCU-CHEK test strips (Roche Diagnos-

tics, Indianapolis, IN, USA). The blood glucose level was measured

3 weeks after STZ administration, and only the rats with high blood

glucose level (> 300 mg/dL) were used. Neuropathic pain indiabetic rats was confirmed by examination of nociceptive mechan-

ical thresholds by using the paw pressure Analgesy-Meter (Ugo

Basile Biological Research, Comerio, Italy) (Chen and Pan 2002,

2006).

Isolation of DRG neurons

Rats were anesthetized with 23% isoflurane and then rapidly

decapitated. The thoracic and lumbar segments of the vertebral

column were surgically removed. The lumbar DRGs and the nerve

roots were quickly dissected out and transferred immediately into

DMEM (Gibco, Carlsbad, CA, USA) on ice. The DRGs were then

dissected free of the attached connective tissues under a microscope

and minced with fine-spring scissors. The minced ganglion

fragments were placed in a flask containing 5 mL of DMEM inwhich trypsin (type I, 0.2 mg/mL; Sigma) and collagenase (type I,

1 mg/mL; Sigma) had been dissolved. After incubation at 34C in a

shaking water bath for 40 min, soybean trypsin inhibitor (type II,

1.25 mg/mL; Sigma) was added to terminate the digestion. The cell

suspension was subsequently plated onto a 35-mm culture dish

containing poly-L-lysine (50 lg/mL) pre-coated coverslips and

incubated in 5% CO2 at 37C for 1 h. The supernatant was then

removed, and fresh DMEM was carefully added. The cells were

then kept in the incubator for at least another hour before they were

used for electrophysiological recordings. The final electrophysio-

logical recordings were performed 26 h after dissociation to allow

recovery from trypsination and mechanical disruption.

Electrophysiological recordings

The electrodes were pulled from GC150TF-10 glass capillaries

(inner diameter, 1.17 mm; outer diameter, 1.5 mm; Harvard

Apparatus, Holliston, MA, USA) using a micropipette puller and

fire-polished. The neurons were visualized using differential

interference contrast optics on an inverted microscope (Olympus,

Tokyo, Japan). Images of the cells were taken with a CCD camera

and displayed on a video monitor. The neurons were recorded in the

whole-cell configuration at a holding potential of)90 mV using an

EPC-10 amplifier (HEKA Instruments, Lambrecht, Germany). After

the whole-cell configuration was established, the cell membrane

capacitance and series resistance were electronically compensated.

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Leak currents were subtracted using the online P/4 protocol. All

experiments were performed at 25C. Signals were filtered at

1 kHz, digitized at 10 kHz, and acquired using the Pulse software

program.

To selectively record Kv currents and minimize the contribution

from Ca2+and Na+ currents, the extracellular solution contained (in

mM) 150 choline chloride, 5 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES,

1 CdCl2, and 10 D-glucose (pH 7.4 adjusted with KOH, osmolarity

320 mOsm). Because both 4-AP and DAP can directly stimulate

high voltage-gated Ca2+channels (Wu et al. 2009), CdCl2 was used

to block high voltage-gated Ca2+channels. The electrode resistance

was 23 MW when filled with the solution containing (in mM) 120

potassium gluconate, 20 KCl, 2 MgCl2, 10 EGTA, 10 HEPES, 5

Na2-ATP, and 1 CaCl2 (pH 7.2 adjusted with KOH, osmolarity

300 mOsm). To further determine the Kv current subtype in the

DRG neurons, the IA and IK currents were differentiated using

25 mM TEA and 5 mM DAP, blockers of native IK and IA,

respectively (Robertson and Nelson 1994; Safronov et al. 1996;

Everill et al. 1998; Vydyanathan et al. 2005). DAP was used in thisstudy because it shows a more potent inhibition of IA than 4-AP

(Robertson and Nelson 1994). The protocol used to measure Kv

current activation was performed at a holding potential of)90 mV

and consisted of 400-ms depolarization pulses from )70 to 60 mV

in 10-mV increments at 2-s intervals (Vydyanathan et al. 2005).

Drug application

The drugs were dissolved in distilled water at 1000 times the final

concentration and kept frozen in aliquots. The stock solutions were

diluted in the appropriate external solution just before use and held

in a series of independent syringes connected to corresponding fused

silica columns (ID 200 lm). The distance from the column mouth to

the cell being recorded was about 100 lm. Each drug solution was

delivered to the recording chamber by gravity, and rapid solutionexchange (about 200 ms) was achieved by controlling the corre-

sponding valve switch. All drugs and chemicals were purchased

from Sigma-Aldrich except BDNF and the anti-BDNF antibody,

which were purchased from Millipore (Temecula, CA, USA).

Real-time RT-PCR analysis of Kv subunit expression

Total RNA was extracted from rat lumbar DRGs at the L4-L6 level

using the Purelink total RNA purification system (Invitrogen,

Carlsbad, CA, USA) with on-column Dnase I digestion according to

the manufacturers instructions. cDNA was prepared by using the

Superscript III first-strand synthesis kit (Invitrogen).

Quantitative PCR was performed using the iQ5 real-time PCR

detection system with the SYBR Green PCR kit (Bio-Rad,

Hercules, CA, USA). All samples were analyzed in duplicate using

an annealing temperature of 60C, and each experiment was

repeated at least once. The primer pairs used are listed in Table 1.

To calculate the relative Kv subunit mRNA expression levels in

each sample, standard curves were generated using a twofold

dilution of the cDNA from the DRGs as the PCR template. The

relative amount of Kv-subunit mRNA in each sample was first

normalized to the level of the housekeeping gene S18 and was then

normalized to its expression level in control rats. The PCR product

specificity was verified by melting-curve analysis and agarose gel

electrophoresis.

Double immunofluorescence labeling of BDNF and Nissl in the DRG

Four rats in each group were used for the immunofluorescence

labeling. Rats were used 3 weeks after treatment with either STZ or

vehicle control. Under deep anesthesia induced by sodium pento-

barbital (60 mg/kg, i.p.), rats were perfused intracardially with

250 mL of saline, 250 mL of 4% paraformadehyde in 0.1 M

phosphate-buffered saline (PBS), and 150 mL of 10% sucrose in

PBS (pH 7.4). The lumbar DRGs at the L4-L6 levels were removed

quickly and cryoprotected in 30% sucrose in PBS for 24 h at 4C.

To determine the distribution of BDNF in DRG neurons in both

control and diabetic rats, immunofluorescent labeling of BDNF and

Nissl (a neuronal marker) in the DRG sections were performed. The

DRG tissues were cut into 30-lm-thick sections and collected free-floating in 0.1 M PBS. The tissue sections were rinsed in 0.1 M

Tris-buffered saline, and incubated with the primary antibody (rabbit

anti-BDNF, dilution 1 : 500; Millipore) for 2 h at 25C and then

5 days at 4C. The specificity of this antibody has been demon-

Table 1 List of primers used for real-time PCR

Gene name Primer Sequence Location

rat Kv1.1 Kv1.1-p1 5 gga gcg ccc cct acc cga gaa g 3 454475

(NM_173095) Kv1.1-p2 5 ggt gaa tgg tgc ccg tga agt cct 3 644621

rat Kv1.2 Kv1.2-p1 5 tcc cgg atg cct tct ggt g 3 16631683

(NM_012970) Kv1.2-p2 5

ggc ctg ctc ctc tcc ctc tgt 3

18621842rat Kv1.4 Kv1.4-p1 5 ttg tga acg cgt ggt aat aaa tgt gt 3 605630

(NM_012971) Kv1.4-p2 5 ggc ggc ctc ctg act ggt aat aat a 3 804780

rat Kv2.1 Kv2.1-p1 5gcg act gct cag acc cct tag ctc 3 239262

(NM_013186) Kv2.1-p2 5 tct gga atc gtg atc agc gct ttg 3 11361113

rat Kv2.2 Kv2.2-p1 5 cgt gga gaa ggc tgg aga gtc g 3 17271748

(NM_054000) Kv2.2-p2 5 tgg gct gga gga aga agt gtt gtt 3 19191896

rat Kv3.4 Kv3.4-p1 5 cca cgg ggc aat gac cac acc 3 643663

(XM_001070801) Kv3.4-p2 5 aca cag cgc acc cac cag cat tcc t 3 777753

rat Kv4.2 Kv4.2-p1 5 gcc gca gcg cct agt cgt tac c 3 12981319

(NM_031730) Kv4.2-p2 5 tga tag cca ttg tga ggg aaa aga gca 3 15591533

rat Kv4.3 Kv4.3-p1 5 ctc cct aag cgg cgt cct ggt cat t 3 12531277

(NM_031739) Kv4.3-p2 5 ctt ctg tgc cct gcg ttt atc tgc tct c 3 13611334


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strated previously by pre-adsorption with the immunizing antigens

(Zhou and Rush 1996; Zhou et al. 1999). The sections were rinsed

in Tris-buffered saline and incubated with the secondary antibody

(biotin-conjugated goat anti-rabbit IgG, dilution 1 : 200; Jackson

ImmunoResearch Inc., West Grove, PA, USA) for 2 h at 25C.

Then, the sections were incubated with a 1 : 100 dilution of

streptavidin-horseradish peroxidase for 1 h at25C. Subsequently,

the sections were incubated with fluorescein-tyramide (dilution

1 : 100; PerkinElmer, Waltham, MA, USA) for 10 min at 25C.

The sections were then rinsed and incubated with NeuroTrace red

fluorescent Nissl stain (dilution 1 : 100; Molecular Probes, Eugene,

OR, USA) for 40 min at25C. Finally, the sections were rinsed,

Fig. 1 Reduction in the current densities of total Kv, 3,4-diamino-

pyridine (DAP)-sensitive IA, and tetraethylammonium (TEA)-sensitive

IK in medium dorsal root ganglion (DRG) neurons from diabetic rats.

(a, b) Representative traces showing different types of Kv currents in

medium DRG neurons from a control and diabetic rat. The neurons

were held at )90 mV and depolarized from )70 to 60 mV in 10-mV

increments (inset). (c) IV curves show the current densities of total

Kv, IA, and IK in medium DRG neurons from rats in the control ( n = 22

cells) and diabetic (n = 24 cells) groups. (d) Voltage-dependent

activation kinetics (GV curves) of total Kv, IA, and IK in medium

DRG neurons from control and diabetic rats. The V0.5 values of total

Kv currents (control rats: 2.77 2.11 mV, n = 22; diabetic rats:

10.57 1.71 mV, n = 25, p < 0.05) and IK (control rats: 4.85

2.43 mV, n = 17; diabetic rats: 13.92 3.20 mV, n = 20, p < 0.05),

but not IA (control rats: 10.18 5.59 mV, n = 19; diabetic rats: 1.35

4.51 mV, n = 22, p > 0.05), were significantly different between the

control and diabetic rats (t-test). There was no significant difference in

the k value of total Kv currents (control rats: 17.75 0.75, n = 22;

diabetic rats: 19.39 0.70, n = 25, p > 0.05), IA (control rats:

18.10 2.21, n = 19; diabetic rats: 16.09 0.82, n = 22, p > 0.05),

and IK (control rats: 15.05 0.59, n = 17; diabetic rats: 16.45 0.61,

n = 20, p > 0.05) between the control and diabetic rats (t-test).

*p < 0.05 compared with the corresponding value in the control group

(two-way ANOVA).

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mounted on slides, dried, and sealed with a coverslip. The negative

controls were processed in the same manner except the primary

antibody was omitted. The sections were examined on a laser

scanning confocal microscope (Carl Zeiss, Jena, Germany), and the

areas of interest were photographed. To quantify the changes in the

BDNF-expressing neurons from the DRG of diabetic rats, the

number of BDNF-immunoreactive neurons and Nissl-positive

neurons in the DRG of control and diabetic rats was counted by

an investigator who was blinded to the experimental groups. The

cell counting was performed using three images per tissue section,

and three tissue sections were randomly selected from the L5 DRG

for each rat (n = 4 rats per group).

(a)

(b)

(c)

(d)

Fig. 2 Reduction in the current densities of total Kv, 3,4-diamino-

pyridine (DAP)-sensitive IA, and 3,4-diaminopyridine (TEA)-sensitive

IK in large dorsal root ganglion (DRG) neurons from diabetic rats. (a, b)

Original traces show different types of Kv currents in large DRG

neurons from a control and diabetic rat. Neurons were held at )90 mV

and depolarized from )70 to 60 mV in 10-mV increments (inset). (c)

IVcurves show differences in the current densities of total Kv, I A, and

IK in large DRG neurons between the control (n = 19 cells) and dia-

betic (n = 17 cells) groups. (d) Voltage-dependent activation kinetics

of total Kv, IA, and IK in large DRG neurons from control and diabetic

rats. The V0.5 values of total Kv currents (control: 1.38 2.21 mV,

n = 19; diabetic: 11.98 2.66 mV, n = 17, p < 0.05) and IK (control:

4.67 2.57 mV, n = 10; diabetic: 17.16 3.71 mV, n = 11, p < 0.05),

but not IA (control:)4.71 3.02 mV, n = 19; diabetic: 3.33 3.97 mV,

n = 17, p > 0.05), were significantly different between control and

diabetic rats (t-test). There was no significant difference in the kvalue

of total Kv currents (control: 19.66 0.95, n = 19; diabetic:

20.47 0.75, n = 17, p > 0.05), IA (control: 16.51 1.56, n = 19;

diabetic: 13.25 0.84, n = 17, p > 0.05), and IK (control: 16.93 1.06,

n = 10; diabetic: 20.19 2.15, n = 11, p > 0.05) between the two

groups (t-test). *p < 0.05 compared with the corresponding value in

the control group (two-way ANOVA).


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Data analysis and curve fitting

Data are presented as mean SEM. The electrophysiological data

were analyzed using the PulseFit software program (HEKA). The

amplitude of the total Kv currents was measured at the peak, and the

amplitude of the DAP-sensitive IA and TEA-sensitive IK were

obtained by subtracting the amplitude of the DAP- and TEA-

resistant Kv currents from that of the total Kv currents, respectively.

The whole-cell currentvoltage (IV) curves for individual neurons

(a)

(b)

(c)

(d)

Fig. 3 Lack of changes in the total Kv, 3,4-diaminopyridine (DAP)-

sensitive IA, and tetraethylammonium (TEA)-sensitive IK in small dorsal

root ganglion (DRG) neurons from diabetic rats. (a, b) Current traces

showing different types of Kv currents in small DRG neurons from a

control and diabetic rat. Neurons are held at )90 mV and depolarized

from )70 to 60 mV in 10-mV increments (inset). (c) Comparison of the

current densities of total Kv currents, IA, and IK in small DRG neurons

between the control (n = 41 cells) and diabetic (n = 39 cells) group. (d)

Steady-state activation (GV) curvesof total Kv, IA,andIK in small DRG

neurons from control and diabetic rats. There was no significant dif-

ference in the V0.5 value of total Kv currents (control: 3.80 1.98 mV,

n = 36; diabetic: 1.01 2.02 mV, n = 39, p > 0.05), IA (control:

10.40 4.99 mV, n = 36; diabetic: 8.58 3.81 mV, n = 37, p > 0.05),

and IK (control: 17.29 3.06 mV, n = 23; diabetic: 13.12 2.43,

n = 15, p > 0.05) between the control and diabetic rats ( t-test). Also,

the k values showed no difference in total Kv currents (control:

16.22 0.69, n = 36; diabetic: 17.01 0.71, n = 39, p > 0.05), IA

(control: 15.29 1.55, n = 36; diabetic: 16.85 1.47, n = 37,

p > 0.05), and IK (control: 17.53 2.31 mV, n = 23; diabetic:

17.49 1.42, n = 15, p > 0.05) between the two groups (t-test).

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were generated by calculating the peak outward current at each

testing potential and normalizing to the cell capacitance. The

conductancevoltage (GV) curves were described with the Boltz-

mann equation: G/Gmax = 1/[1 + exp(V0.5 ) Vm/k)], where V0.5 is

the membrane potential at which 50% activation is observed, kis the

slope of the function, and Vm is the membrane potential. Differences

between the means were tested for significance using paired or

unpaired Students t-tests, repeated-measures ANOVA followed by

Dunnetts post hoc test, or two-way ANOVA followed by Bonferronis

post hoc test. A p-value of < 0.05 was considered to be statistically

significant.

Results

Three weeks after diabetic induction, the diabetic rats

showed a large reduction in their paw withdrawal thresh-

olds in response to the pressure stimulus applied to the

hindpaw (Chen and Pan 2002; Chen et al. 2009), as

compared with age-matched control rats (control rats:

121.11 3.51 g; diabetic rats: 77.78 3.64 g, p < 0.05,

t-test).

The DRG neurons were divided into three groups

according to their cell diameters, which were measured

with a calibrated eyepiece reticule: small (< 30 lm),

medium (3040 lm), and large (> 40 lm). To determine

the whole-cell Kv currents in these three groups of DRG

neurons, we normalized the peak outward current to the

cell capacitance. There were no significant differences in

the capacitance of the three groups of DRG neurons

between the diabetic rats (small, 26.23 1.08 pF, n = 39;

medium, 62.98 3.75 pF, n = 24; large, 114.51 6.35 pF,

n = 17) and age-matched control rats (small, 25.77

1.99 pF, n = 41; medium, 60.33 2.42 pF, n = 22; large,

121.94 7.36 pF, n = 19; p < 0.05, two-way ANOVA).

Fig. 4 Lack of differences in the total Kv,

3,4-diaminopyridine (DAP)-sensitive IA, and

tetraethylammonium (TEA)-sensitive IK in

isolectin B4 (IB4)-positive and IB4-negative

small dorsal root ganglion (DRG) neurons

between the control and diabetic group. (a,

b) IV curves show the similar amplitudes

of the Kv current density in IB4-positive and

IB4-negative neurons at different potentialsin the control (IB4-positive, n = 13 cells; IB4-

negative, n = 5 cells) and diabetic (IB4-po-

sitive, n = 31 cells; IB4-negative, n = 5

cells) groups.

Fig. 5 Changes in the mRNA levels of individual Kv subunits in the

dorsal root ganglion (DRG) from diabetic rats. (a) Differences in the

mRNA levelsof IA subunits(Kv1.4,Kv3.4, Kv4.2, andKv4.3) in the DRG

between control and diabetic rats. (b) Lack of differences in the mRNA

levels of IK subunits (Kv1.1, Kv1.2, Kv2.1, and Kv2.2) in the DRG

between control and diabetic rats (n = 8 rats, in each group). *p < 0.05

compared with the corresponding value in the control group (t-test).


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(a)

(b)

(c)

(d)

(e)

Fig. 6 Effects of brain-derived neurotrophic

factor (BDNF) treatment on the total Kv,3,4-diaminopyridine (DAP)-sensitive IA, and

tetraethylammonium (TEA)-sensitive IK

currents in dorsal root ganglion (DRG)

neurons from control rats. (a) IV curves

show that BDNF treatment had no effect on

the current densities of total Kv, IA, and IK in

small DRG neurons (n = 12). (b) IVcurves

show that BDNF treatment reduced the

current densities of total Kv, IA, and IK in

medium DRG neurons (n = 13). (c) IV

curves show that reduced the current den-

sities of total Kv, IA, and IK in large DRG

neurons (n = 12). (d) K252a, but not K252b,

abolished the BDNF effects on total Kv, IA,

and IK in medium DRG neurons (n = 7 in

each group). (e) K252a, but not K252b,

blocked the BDNF effects on total Kv, IA,

and IK in large DRG neurons (n = 8 in each

group). *p < 0.05 compared with the corre-

sponding value in the control or vehicle

(K252b) group (two-way ANOVA). [Correction

after online publication 27 July 2010: Figure

6 was replaced with the correct version of

the figure.]

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Reduction in Kv currents in medium and large DRG neurons

from diabetic rats

The total Kv current density was significantly reduced in

diabetic rats in both medium and large DRG neurons, as

compared with the total Kv current density in the DRGneurons from the control group (Figs 1 and 2). The peak

current density of both the IA and IK in the medium and large

DRG neurons was also significantly smaller in the diabetic

than in the control group (Figs 1 and 2).

Steady-state activation is an important property of Kv

currents and can influence the excitability of DRG neurons.

Thus, we determined the steady-state activation of total Kv,

IA, and IK in medium and large DRG neurons. There was a

significant depolarizing shift in the total Kv and IK currents

in medium DRG neurons from diabetic rats (total Kv,

V0.5 = 10.57 1.71 mV; IK, V0.5 = 13.92 3.20 mV), as

compared with those from the control rats (total Kv,

V0.5 = 2.77 2.11 mV; IK, V0.5 = 4.85 2.43 mV; t-test)

(Fig. 1d). Also, a similar depolarizing shift was found in

the IK in the large DRG neurons from the diabetic rats (total

Kv, V0.5 = 11.98 2.66 mV; IK, V0.5 = 17.16 3.71 mV),

as compared with those from the control rats (total Kv,

V0.5 = 1.38 2.21 mV; IK, V0.5 = )4.67 2.57 mV; t-test)

(Fig. 2d). However, there was no significant difference in the

steady-state activation of IA in the medium and large DRG

neurons between the diabetic and control groups (Figs 1dand 2d).

Lack of changes in Kv currents in small DRG neurons in

diabetic rats

The current densities of the total Kv, IA, and IK in small DRG

neurons from diabetic rats were not significantly different

from the current densities in small DRG neurons from control

rats (Fig. 3). In addition, the activation kinetics of the total,

IA, and IK from small DRG neurons did not differ signifi-

cantly between the control and diabetic rats (Fig. 3d).

Because the phenotypes of the small DRG neurons are

heterogenous, we further examined the Kv currents in IB4-

positive and IB4-negative small DRG neurons in diabetic and

control rats. Immediately before recording, the neurons were

labeled with IB4Alexa Fluor 594 (3 lg/mL) in a Tyrode

solution for 10 min and then rinsed for at least 3 min

Fig. 7 Effects of the anti-brain-derived

neurotrophic factor (BDNF) antibody on the

current densities of total Kv, 3,4-diamino-

pyridine (DAP)-sensitive IA, and tetraethy-

lammonium (TEA)-sensitive IK currents in

dorsal root ganglion (DRG) neurons from

diabetic rats. (a) IV curves show that

treatment with the anti-BDNF antibody

(50 ng/mL) slightly increased the current

densities of total Kv, IA, and IK in small DRG

neurons (n = 8). (b) IV curves show that


profoundly increased the current densities

of total Kv, IA, and IK in medium DRG neu-

rons (n = 13). (c) IV curves show that


substantially increased the current densities

of total Kv, IA, and IK in large DRG neurons

(n = 9). Note that the boiled anti-BDNF

antibody had no effects on the Kv current

density in small, medium, or large DRG

neurons. *p < 0.05 compared with the cor-

responding value in the control group (two-

way ANOVA).


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(Vydyanathan et al. 2005). There were no significant differ-

ences in the Kv current densities in the IB4-positive or IB4-

negative neurons between the control and diabetic groups

(Fig. 4).

Changes in mRNA levels of IA and IK subunits in the DRG

from diabetic rats

We next measured the mRNA levels of Kv a-subunits in the

DRG from control and diabetic rats. Because the Kv1.4,

Kv3.4, Kv4.2, and Kv4.3 subunits contribute to the IA

channels in DRG neurons (Stuhmer et al. 1989; Oliveret al.

2004; Chien et al. 2007), we measured the mRNA levels of

Kv1.4, Kv3.4, Kv4.2, and Kv4.3 in the DRG from control

and diabetic rats. The mRNA levels of the Kv1.4, Kv3.4,

Kv4.2, and Kv4.3 subunits were all reduced approximately

50% in the diabetic rats, as compared with the levels in the

control rats (Fig. 5a). Kv1.1, Kv1.2, Kv2.1, and Kv2.2 are

important subunits of the IK channels (Murakoshi and

Trimmer 1999; Malin and Nerbonne 2002; Beekwilder et al.

2003). However, there were no significant differences in the

mRNA levels of the Kv1.1, Kv1.2, Kv2.1, and Kv2.2

subunits in the DRG between the control and diabetic rats

(Fig. 5b).

Role of BDNF in diabetes-induced reduction in Kv currents

in medium and large DRG neuronsIt has been shown that BDNF expression is increased in the

rat DRG after diabetic induction (Fernyhough et al. 1995).

To determine whether increased BDNF contributes to the

reduction in Kv currents in the DRG in diabetic neuropathy,

we first determined whether BDNF treatment affects Kv

currents in DRG neurons from control rats. Treatment of

DRG neurons from control rats with 50 ng/mL of BDNF for

24 h (Youssoufian and Walmsley 2007) significantly

reduced the peak current densities of the total, IA, and IK

in medium- and large-diameter neurons (Fig. 6ac). How-

ever, BDNF treatment had no significant effect on the Kv

current density in the small DRG neurons from control rats.

TrkB is the high-affinity BDNF receptor and is primarily

present in medium and large DRG neurons (McMahon et al.

1994; Wetmore and Olson 1995; Karchewski et al. 1999).

We next determined whether the effect of BDNF on Kv

Fig. 8 Effects of K252a on the current

densities of total Kv, 3,4-diaminopyridine

(DAP-sensitive) IA, and tetraethylammoni-

um (TEA)-sensitive IK currents in dorsal root

ganglion (DRG) neurons from diabetic rats.

(a) IV curves show that treatment with

K252a (300 nM) slightly increased the cur-

rent densities of total Kv, IA, and IK in small

DRG neurons (n = 11). (b) IVcurves show

that treatment with K252a substantially in-

creased the current densities of total Kv, IA,

and IK in medium DRG neurons (n = 10).

(c) IV curves show that treatment with

K252a profoundly increased the current

densities of total Kv, IA, and IK in large DRG

neurons (n = 9). *p < 0.05 compared with

the corresponding value in the diabetic

control group (two-way ANOVA).

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currents in DRG neurons was mediated by TrkB. Pre-

treatment of DRG neurons with K252a (300 nM) (Tapley

et al. 1992; Bhave et al. 1999), a TrkB receptor inhibitor,

blocked the BDNF effect on the Kv current density in DRG

neurons from control rats (Fig. 6d and e). The inactive

analogue of K252a, K252b (300 nM), did not significantly

alter the BDNF effect on the Kv current density in these

neurons.

To examine the role of BDNF in the diabetes-induced

reduction in the Kv current density, we next tested the effect

of the anti-BDNF antibody on the Kv currents in DRG

neurons from diabetic rats. Incubation of DRG neurons from

diabetic rats with the BDNF antibody (1 : 50) for 24 h

caused a large increase in the peak current densities of the

total Kv in all sizes of DRG neurons (Fig. 7). Also, there was

a large increase in the IA and IKdensities in medium and large

DRG neurons after treatment with the anti-BDNF antibody

(Fig. 7b and c). Treatment with the boiled BDNF antibody

had no effect on the Kv current density in DRG neurons.

To test the hypothesis that BDNF reduces the Kv

currents through TrkB receptor stimulation in diabetic

neuropathy, we assessed the effect of K252a on the Kv

current density of DRG neurons from diabetic rats.

Incubation of DRG neurons with K252a (300 nM), for

24 h profoundly increased the total Kv current density in

all sizes of DRG neurons from diabetic rats (Fig. 8).

Treatment with K252a also increased the IA and IK

densities in all three groups of DRG neurons from diabetic

rats (Fig. 8).

Effects of anti-BDNF antibody and K252a on Kv currents in

DRG neurons from control rats

To estimate whether BDNF and its receptors (TrkB) are up-

regulated in DRG neurons in diabetic neuropathy, we also

examined the effects of anti-BDNF antibody and K252a on

Kv current density in DRG neurons from control rats. In

contrast to the evident effects of anti-BDNF antibody and

K252a on Kv currents of DRG neurons from diabetic rats,

treatment with BDNF antibody (1 : 50) or K252a

(300 nM), for 24 h had little effects on the Kv current

density in all sizes of DRG neurons obtained from control

rats (Fig. 9).

Fig. 9 Effects of K252a and anti-brain-

derived neurotrophic factor (BDNF) anti-

body on the current densities of total

Kv, 3,4-diaminopyridine (DAP)-sensitive IA,

and tetraethylammonium (TEA)-sensitive Ik

currents in dorsal root ganglion (DRG)neurons from control rats. (a) IV curves

show that treatment with K252a (300 nM,

n = 11) and anti-BDNF antibody (50 ng/mL,

n = 9) only had a small effect on the current

density of IA in small DRG neurons. (b) IV

curves show the effect of treatment with

K252a (n = 9) and anti-BDNF antibody

(n = 10) on the current densities of total Kv,

IA, and Ik in medium DRG neurons. (c) IV

curves show the lack of effect of treatment

with K252a (n = 8) and anti-BDNF antibody

(n = 8) on the current densities of total Kv,

IA, and Ik in large DRG neurons. *p < 0.05

compared with the corresponding value in

the control group (two-way ANOVA).


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Role of BDNF in diabetes-induced reduction in the

expression levels of IA subunits in DRG neurons

Because we found a large reduction in the mRNA levels of IA

subunits in the DRG tissue of diabetic rats, we further

determined the role of BDNF in diabetes-induced decreases

in the expression level of Kv1.4, Kv3.4, Kv4.2, and Kv4.3

subunits in DRG neurons using the real-time PCR technique.

Incubation of BDNF (50 ng/mL) for 4 h in DRG neurons

from control rats caused a large decrease in the mRNA levels

of Kv1.4, Kv3.4, Kv4.2, and Kv4.3 subunits (Fig. 10a).

Furthermore, in DRG neurons from diabetic rats, treatment

with the anti-BDNF antibody (1 : 50) or K252a (300 nM)

for 4 h reversed the decrease in the mRNA levels of Kv1.4,

Kv3.4, Kv4.2, and Kv4.3 subunits (Fig. 10b).

Altered distribution patterns of BDNF-immunoreactive

DRG neurons in diabetic rats

Additionally, we determined whether diabetic neuropathy

affects the distribution of BDNF in different sized DRG

neurons. In control rats, BDNF immunoreactivity was

distributed in some small and medium DRG neurons. In

contrast, BDNF immunoreactivity was present in most

medium and large DRG neurons from diabetic rats

(Fig. 11a). The total number of BDNF-immunoreactive

neurons in the DRG was much greater in the diabetic

(2487/4808, 51.72%) than in the control (1108/5040,21.98%; chi-squared test) rats. There were more medium

and large neurons with BDNF immunoreactivity in the DRG

from diabetic rats than control rats (Fig. 11b).

Discussion

In the present study, we found that the density of Kv currents,

especially the IA, in medium and large DRG neurons was

significantly reduced in a rat model of painful diabetic

neuropathy. Quantitative PCR analysis showed that the

mRNA levels of IA subunits, including Kv1.4, Kv3.4, Kv4.2,

and Kv4.3, were significantly reduced in the DRG of diabetic

rats. However, there were no significant differences in the

mRNA levels of the IK subunits (Kv1.1, Kv1.2, Kv2.1, and

Kv2.2) in the DRG between the diabetic and control rats.

Furthermore, the large reduction in the Kv current density

observed in the diabetic rats was mimicked by treatment of

DRG neurons from control rats with BDNF. Treatment with

either the anti-BDNF antibody or a TrkB inhibitor reversed

the changes in the Kv current density of DRG neurons from

diabetic rats but had little effect on Kv currents in all sizes of

DRG neurons from control rats. In addition, the number of

medium and large DRG neurons with BDNF immunoreac-

tivity was markedly increased in diabetic rats. Therefore, our

parallel biochemical and electrophysiological results provideimportant new information that diabetic neuropathy reduces

Kv activity, particularly the IA, in medium and large DRG

neurons. Increased BDNF activity likely contributes to the

reduction in the Kv current density through TrkB receptor

stimulation in diabetic neuropathy.

Kv channels are crucial in the control of neuronal

excitability, and their down-regulation can increase neuronal

excitability (Pongs 1999; Vydyanathan et al. 2005; Chi and

Nicol 2007; Chien et al. 2007; Catacuzzeno et al. 2008).

We found here that there was a profound decrease in the

density of total Kv, IA, and IK in medium and large DRG

neurons from diabetic rats. In addition, the reduction in the

IA density was greater than the reduction in the IK density in

these DRG neurons from diabetic rats. Consistent with our

electrophysiological data, the mRNA levels of IA subunits,

including Kv1.4, Kv3.4, Kv4.2, and Kv4.3, were signifi-

cantly reduced in the DRG from diabetic rats. Thus, reduced

expression of IA subunits in diabetes could account for the

reduced IA density seen in the medium and large DRG

neurons from diabetic rats. Inhibition of the IA can increase

the firing frequency and broadening of the action potential,

leading to increased Ca2+influx and neurotransmitter release

(Hoffman et al. 1997; Vydyanathan et al. 2005; Catacuzz-

eno et al. 2008). For example, knockout of the Kv4.2

Fig. 10 Reduction in the mRNA levels of IA subunits by brain-derived

neurotrophic factor (BDNF) in dorsal root ganglion (DRG) neurons. (a)

Effects of BDNF treatment on the mRNA levels of IA subunits (Kv1.4,

Kv3.4, Kv4.2, and Kv4.3) in DRG neurons from control rats (n = 4

samples in each group; t-test). (b) Effects of treatment with the anti-

BDNF antibody (50 ng/mL) or K252a (300 nM) on the mRNA levels of

Kv1.4, Kv3.4, Kv4.2, and Kv4.3 subunits in DRG neurons from diabetic

rats (n = 5 samples in each group; repeated measures ANOVA).*p < 0.05 compared with the corresponding value in the control group.

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subunit reduces the IA and increases the excitability of DRG

neurons, resulting in enhanced sensitivity to tactile and

thermal stimuli (Hu et al. 2006). Furthermore, down-

regulation of IA subunits in DRG neurons induces mechan-

ical hypersensitivity (Chien et al. 2007). It has been shown

that nerve ligation injury decreases the mRNA levels of the

Kv1.1, Kv1.2, Kv1.4, Kv2.2, and Kv4.2 subunits in DRG

neurons (Rasband et al. 2001; Kim et al. 2002). However,

we found that although the mRNA level of Kv1.4 was

significantly reduced in the diabetic group, the mRNA levels

of Kv1.1 and Kv1.2 did not differ significantly between the

control and diabetic groups. This discrepancy is likely

caused by the difference in the peripheral nerve damage

caused by traumatic nerve ligation and diabetic neuropathy.

Data from our present study suggest that the reduced IA in

medium and large DRG neurons could result from reduced

expression of IA subunits and contributes to the abnormal

hyperexcitability of DRG neurons in diabetic neuropathic

pain.

IK channels shape action potentials by keeping the single

action potential short and elevating the firing adaptation

(Safronov et al. 1996; Lien and Jonas 2003; Catacuzzeno

et al. 2008). For instance, suppression of Kv1.1 by dendro-

toxin-K or siRNA enhances the firing activity of DRG

neurons (Chi and Nicol 2007). In addition, Kv1.1 mutant

mice show increased pain responses (Clark and Tempel

1998). Thus, reduction of IK channels may also contribute to

the abnormal hyperexcitability of DRG neurons in painful

diabetic neuropathy. We found that the IK density was

reduced mainly in the medium and large DRG neurons from

diabetic rats. We also observed a depolarizing shift in the IK

in these neurons, which suggests that it is unfavorable to

open IK channels on primary sensory neurons in diabetic

neuropathy. However, the mRNA levels of the IK subunits,

including Kv1.1, Kv1.2, Kv2.1, and Kv2.2, did not differ

significantly between the control and diabetic rats. The

mechanisms underlying the reduction in the IK in DRG

neurons in diabetic neuropathy are not clear. The depolar-

izing shift in the IK alone is not sufficient to explain the large

reduction in the IK density in the medium and large DRG

neurons from diabetic rats. Post-translational regulation,

such as phosphorylation, may play a role in this reduction in

the IK density in DRG neurons in diabetic rats. For example,

the Kv channel activity reconstituted by Kv1.1 or Kv2.1 is

controlled by phosphorylation (Boland and Jackson 1999;

Park et al. 2006). It has been shown that diabetes increases

the activity of protein kinase C (Ishii et al. 1998) and that

increased protein kinase C activation can inhibit Kv1.1

(a)

(b)

Fig. 11 Differences in the distribution pat-

tern of brain-derived neurotrophic factor

(BDNF) immunoreactive neurons in the

dorsal root ganglion (DRG) between control

and diabetic rats. (a) Representative con-

focal images show a greater number of

BDNF immunoreactive neurons in mediumand large neurons from a diabetic rat. Col-

ocalization of BDNF and Nissl (a neuronal

marker) is indicated in yellow when the two

images are digitally merged. Images are

single confocal optical sections. (b) The

histogram shows the distinct differences in

the distribution of BDNF immunoreactive

neurons in different sized DRG neurons

between control and diabetic rats. *p < 0.05

compared with the corresponding value for

the non-treated diabetic group (chi-squared

test).


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(Boland and Jackson 1999). Thus, increased protein kinase C

activity in DRG neurons could reduce the IK density through

phosphorylation of certain IK subunits in diabetic neuro-

pathy.

Previous studies of sensory neurons in diabetic neuro-pathic pain have largely focused on small DRG neurons,

because normal nociception is thought to be mediated

primarily by small-sized DRG neurons and unmyelinated

afferent fibers. We have shown previously that transient

receptor potential vanilloid 1 receptors (TRPV1)-expressing

small sensory neurons are not involved in the development

of allodynia in this rat model of diabetic neuropathic pain

(Khan et al. 2002). In addition, damage to large myelinated

afferent fibers is well known in diabetic neuropathy

(Ochodnicka et al. 1995). We found that the diabetes-

induced reduction in Kv currents was limited predominantly

to medium and large DRG neurons. These findings provide

further evidence that medium- and large-sized DRG neurons,

which are typically associated with myelinated primary

afferent fibers, are important in the development of diabetic

neuropathic pain. These results indicate that the reduced Kv

current activity in these primary sensory neurons could

contribute to abnormal hyperexcitability of these primary

sensory neurons and the mechanical allodynia seen in

diabetic neuropathy.

Another important finding of this study is that BDNF

plays a critical role in the reduction in the Kv currents in

DRG neurons from diabetic rats. We found that acute BDNF

treatment reduced the mRNA levels of IA subunits in DRG

neurons. Furthermore, BDNF treatment mainly reduced theKv current density, especially the IA current density, in

medium and large DRG neurons from control rats. The role

of BDNF in the reduction of Kv currents in DRG neurons

from diabetic rats was also supported by our finding that

treatment with the anti-BDNF antibody or a TrkB inhibitor,

K252a, reversed the changes in the Kv currents and the

mRNA levels of IA subunits in DRG neurons from diabetic

rats. Our data suggest that BDNF reduces the IA current

density by inhibiting the expression of IA subunits in DRG

neurons in diabetic rats. Although it remains uncertain how

BDNF suppresses the expression of IA subunits in DRG

neurons, BDNF may inhibit the expression of IA subunits

through the transcriptional repressor neuron restrictive

silencing factor/repressor element-1 Silencing transcription

factor (NRSF/REST). It has been shown that NRSF/REST is

involved in epigenetic silencing of Kv4.3 expression by

nerve injury in DRG neurons (Uchida et al. 2010). It is not

clear to what degree the paradigm of BDNF application used

in this study mimics the BDNF production in the DRG in

diabetes. It has been suggested that the manner of BDNF

application can have a very different effect on functional

outcomes (Greenberg et al. 2009). Because there is no

information about the time course of changes in BDNF

concentrations in the DRG in diabetes, we did not compare

whether slow and rapid increases in BDNF concentrations

produce different effects on Kv currents in DRG neurons. It

should be noted that K252a can block TrkA, TrkB, and

TrkC. It has been shown that treatment with NGF (acting via

TrkA and p75 neurotrophin receptors) can maintain Kvchannel activity after nerve ligation injury in sensory

neurons (Everill and Kocsis 2000). However, because NGF

does not affect the mRNA levels of IA current subunits in

DRG neurons (Park et al. 2003), it is less likely that the

observed effect of K252a on Kv channel activity in diabetic

DRG neurons in this study is mediated by TrkA. We found

that in contrast to the evident effects of anti-BDNF antibody

and K252a on Kv currents of DRG neurons from diabetic

rats, treatment with anti-BDNF antibody or K252a had little

effect on the Kv current density in all sizes of DRG neurons

from control rats. These data suggest that the functional

activity of BDNF is increased, which reduces Kv channel

activity through TrkB receptor stimulation in DRG neurons

of diabetic rats.

In addition, we found that the number of medium and large

DRG neurons that were immunoreactive to BDNF was

increased in diabetic rats. Our findings are consistent with the

results from studies of rats subjected to nerve ligation injury

(Tonra et al. 1998; Zhou et al. 1999). We found that BDNF

treatment had little effect on the Kv currents in small DRG

neurons in control rats. However, the Kv current density in

small DRG neurons was slightly increased after treatment

with the anti-BDNF antibody or a TrkB receptor inhibitor in

the diabetic rats. Because BDNF and TrkB receptors were

normally present in a subpopulation of small DRG neurons(McMahon et al. 1994; Zhou and Rush 1996), it is possible

that BDNF may tonically inhibit the expression of certain Kv

channels through TrkB receptors in these neurons in diabetic

rats. It is not completely clear how BDNF leads to increased

TrkB activation in DRG neurons of diabetic rats. We propose

that BDNF released from diabetic DRG neurons activates

neuronal TrkB through an autocrine mechanism. Therefore,

increased BDNF activity could contribute to the large

reduction in Kv channel function in medium and large

DRG neurons in diabetic neuropathy through augmented

TrkB receptor activation.

In conclusion, this study provides novel information that

diabetic neuropathy reduces Kv currents, particularly the IA,

in medium and large DRG neurons. BDNF likely plays an

important role in the reduction in the Kv channel activity of

these neurons in painful diabetic neuropathy through TrkB

receptor stimulation. Because reduction in Kv currents can

enhance neuronal excitability, increased BDNF activity may

enhance the excitability of DRG neurons by down-regulating

the Kv channels in diabetic neuropathy. This new informa-

tion is important for our understanding of the mechanisms

underlying the hyperactivity of primary sensory neurons and

the increased afferent input to the spinal dorsal horn in

painful diabetic neuropathy.

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Acknowledgements

This study was supported by the National Institutes of Health grants

GM64830 and NS45602 and by the N.G. and Helen T. Hawkins

Endowment to H.L.P.

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