Nociceptor-derived brain-derived neurotrophic factor regulates

acute and inflammatory but not neuropathic pain

Jing Zhao,a,1 Anjan Seereeram,a,1 Mohammed A. Nassar,a Alessandra Levato,a Sophie Pezet,c

Gareth Hathaway,b Cruz Morenilla-Palao,a Caroline Stirling,a Maria Fitzgerald,b

Stephen B. McMahon,c Maribel Rios,d and John N. Wooda,*

London Pain ConsortiumaMolecular Nociception Group, Department of Biology, University College London, London WC1E 6BT, UKbAnatomy and Developmental Biology Department, University College London, London WC1E 6BT, UKcWolfson CARD, The Wolfson Wing, Hodgkin Building, Guy’s Campus, King’s College, London, SE1 1UL, UKdNeuroscience Department, Tufts University Medical School, Boston, MA 02111, USA

Received 10 August 2005; revised 10 November 2005; accepted 17 November 2005

Available online 18 January 2006

Conditional mouse knock-outs provide an informative approach to

drug target validation where no pharmacological blockers exist or

global knock-outs are lethal. Here, we used the Cre-loxP system to

delete BDNF in most nociceptive sensory neurons. Conditional null

animals were healthy with no sensory neuron loss. However, pain-

related behavior was substantially altered. Baseline thermal thresholds

were reduced. Carrageenan-induced thermal hyperalgesia was

inhibited. Formalin-induced pain behavior was attenuated in the

second phase, and this correlated with abolition of NMDA receptor

NR1 Ser896/897 phosphorylation and ERK1 and ERK2 activation in the

dorsal horn; AMPA receptor phosphorylation (GluR1/Ser831) was

unaffected. NGF-induced thermal hyperalgesia was halved, and

mechanical secondary hyperalgesia caused by intramuscular NGF

was abolished. By contrast, neuropathic pain behavior developed

normally. Nociceptor-derived BDNF thus plays an important role in

regulating inflammatory pain thresholds and secondary hyperalgesia,

but BDNF released only from nociceptors plays no role in the

development of neuropathic pain.

D 2005 Elsevier Inc. All right reserved.

Keywords: BDNF; DRG; Conditional knock-outs; Inflammatory pain;

Neuropathic pain; NMDA receptor; Phosphorylation; ERK1; ERK2

Introduction

Brain-derived neurotrophic factor (BDNF) is a member of the

neurotrophin family (Huang and Reichardt, 2003). First implicat-

ed in the survival and maintenance of the peripheral sensory

system during development (Jones et al., 1994; Liu et al., 1995),

BDNF also acts as a regulator of neuronal excitability and

modulator of synaptic plasticity in the central nervous system (Li

et al., 2005; McAllister et al., 1999; Rivera et al., 2004). Evidence

obtained using neutralizing TrkB receptor bodies suggests that

BDNF acts as a neuromodulator when released from small

diameter nociceptive neurons, playing an important role in pain

pathways (Kerr et al., 1999; Thompson et al., 1999). BDNF is

synthesized in the cell bodies of primary sensory neurons and

expressed by a sub-population of small-diameter sensory neurons

with unmyelinated axons that terminate in the superficial laminae

of the dorsal horn (Ernfors et al., 1990). BDNF undergoes

anterograde transport to the dorsal horn where it is associated with

synaptic vesicles of nociceptive neurons and may be released onto

first-order spinal neurons (Michael et al., 1997). BDNF expres-

sion levels in the nervous system are altered in a number of pain

models including peripheral inflammation (Cho et al., 1997a,b),

axotomy and nerve injury and neuropathic pain paradigms (Cho et

al., 1998; Ha et al., 2001; Zhang et al., 2000; Zhou et al., 2000).

TrkB, which is expressed by post-synaptic neurons of the dorsal

horn, is the high affinity receptor for BDNF and NT4. Noxious

stimulation increases the phosphorylation of TrkB in the rat spinal

dorsal horn consistent with the release of BDNF, and this is

associated with increased ERK kinase auto-phosphorylation in the

superficial dorsal horn (Pezet et al., 2002a,b). BDNF also appears

to enhance NMDA-receptor-mediated responses in the dorsal horn

(Kerr et al., 1999; Garraway et al., 2003, 2005). Acute or chronic

noxious stimuli increase the phosphorylation of various NMDA

1044-7431/$ - see front matter D 2005 Elsevier Inc. All right reserved.

doi:10.1016/j.mcn.2005.11.008

* Corresponding author. Molecular Nociception Group, Department of

Biology, University College London, London WC1E 6BT, UK.

E-mail address: J.Wood@ucl.ac.uk (J.N. Wood).1 The first two authors contributed equally to his work.

Available online on ScienceDirect (www.sciencedirect.com).

www.elsevier.com/locate/ymcne

Mol. Cell. Neurosci. 31 (2006) 539 – 548

receptor (NMDAR) subunits in the spinal cord in vivo (Guo et al.,

2002; Brenner et al., 2004). Exogenous BDNF has also been

shown to modulate NR1 phosphorylation (Slack and Thompson,

2002; Slack et al., 2004), although other studies have argued

against an effect of BDNF on NMDA receptor function

(Heppenstall and Lewin, 2001). A role for BDNF in neuropathic

pain has also been proposed. BDNF levels increase in uninjured

DRG neurons after neuropathic insults (Fukuoka et al., 2001).

Antibodies to TrkB or tyrosine kinase inhibitors and TrkB-

neutralizing receptor bodies have been shown to block neuro-

pathic pain (Yajima et al., 2002). On the other hand, a gene

therapy study suggested that exogenous BDNF can have analgesic

effects in neuropathic pain (Eaton et al., 2002). The role of BDNF

in neuropathic pain is thus contentious.

BDNF knock-out animals die during the second postnatal week

precluding a behavioral assessment of the role of BDNF in the

mature sensory system in vivo (Rios et al., 2001). Electrophys-

iological studies of ventral root potentials in p4–p7 BDNF null

mutant mice support a role for BDNF in modulating pain

pathways, consistent with the neutralizing receptor body experi-

ments (Heppenstall and Lewin, 2001; Kerr et al., 1999). However,

the neutralizing receptor bodies may fail to completely sequester

all BDNF and may also sequester NT4; the source of the BDNF

that may regulate synaptic input in the dorsal horn also remains

unknown. The development of a nociceptor-specific Cre strain

(Nav.1.8-Cre) allowed us to generate adult animals deficient in

BDNF in most nociceptive sensory neurons (Stirling et al., 2005).

The aims of this study were to determine the involvement of

nociceptor-derived BDNF in the establishment and maintenance of

a range of behavioral assays including responses to innocuous and

noxious thermal, chemical and mechanical stimuli under both

normal and inflammatory conditions (Kerr et al., 1999; Malcangio

and Lessmann, 2003; Pezet et al., 2002b). Here, we provide

evidence that BDNF released from nociceptors plays a major role

in regulating acute and inflammatory pain but not neuropathic

pain.

Results

Genotyping analysis of floxed BDNF mice and BDNF conditional

null mice

PCR and Southern blotting were used to detect the genotype of

floxed BDNF mice and BDNF conditional null mutants (BDNF

KO). PCR results showed that the floxed BDNF band (440 bp) and

Nav1.8 band (460 bp) were amplified from the tail DNA of floxed

BDNF mice and conditional null mutants. The Cre band (250 bp)

was amplified from the tail DNA of BDNF conditional null

mutants but not floxed BDNF mice (Fig. 1A). Southern blotting

demonstrates that the genotype of BDNF conditional null mutants

is heterozygous Nav1.8-Cre and the genotype of floxed BDNF

mice is homozygous Nav1.8 (Fig. 1B). Assessment of the genotype

ratios gave the expected ratios generated from the breeding

program. Furthermore, the weights of the two groups of females

used for behavioral studies (8–10 weeks post natal) were not

significantly different. Nav1.8-specific BDNF conditional null

mutants 16.88 T 0.88 g compared with wild-type (WT) littermates

17.72 T 0.42 g (P = 0.43; n = 6).

BDNF levels were compared in control and conditional null

mutants by immunocytochemistry and qPCR. Taking the

normalized expression of BDNF in WT mice as 100%, the

expression of BDNF mRNA in the null mutant is about 23% of

this value. This agrees with qualitative immunocytochemical

studies that show a reduction in BDNF-immunoreactive neurons

in sections of DRG taken from null mutants, compared with

controls (Figs. 2A and B).

BDNF null mutants have been shown to contain a normal

complement of DRG neurons (Heppenstall and Lewin, 2001). Not

surprisingly, the same result is true in the conditional null, where

the total number and proportion of neurofilament and peripherin-

positive neurons are identical in wild-type and null littermates

(Figs. 2C and D).

Behavioral studies

Motor coordination measured by means of the Rota-rod test did

not show any significant difference between nulls and littermates

(Fig. 3A). All animals (n = 12) in both groups were able to stay on

the rod, turning at a constant speed of 20 rpm for the maximum

time of 300 s. Subjecting the animals to the increasing speed test

also failed to highlight any differences in time spent on the rotor

rod by the two groups, BDNF null 140.1 T 19.6 s; control

littermates 157.0 T 14.5 s (t test, P = 0.43).

Mechanical pressure thresholds

The Randall – Sellito test, which measures nociceptive

responses to pressure, was applied to the tails of 9 animals of

each genotype (Randall and Sellito, 1957). The means obtained

were BDNF null 68.3 T 4.3 g; control 71.6 T 8.3 g. There is no

significant difference (t test, P = 0.74) in the responses of the

control vs. BDNF KO group (Fig. 3B). Punctate low threshold

mechanical sensitivity was measured using von Frey hairs (Fig.

3C). Again, there were no significant differences (t test, P = 0.59)

between wild-type (0.79 T 0.15 g; n = 9) and BDNF conditional

null mice (0.68 T 0.08 g; n = 7).

Fig. 1. Genotyping analysis of floxed BDNF mice and BDNF conditional

null mice. (A) PCR was used to detect the genotype for floxed BDNF mice

and BDNF conditional null mice. The tail DNA of littermate was amplified

with floxed BDNF primers, Nav1.8 primers and Cre primers. (B) Southern

blotting with BamHI and 3V arm external probe confirms heterozygous

Nav1.8Cre in BDNF conditional null mice and homozygous Nav1.8 in

floxed BDNF mice.

J. Zhao et al. / Mol. Cell. Neurosci. 31 (2006) 539–548540

Thermal thresholds

To assess hyperalgesia or analgesia, a Hargreaves apparatus was

used (Fig. 3D). No differences (t test, P = 0.99; n = 6) in latency to

foot withdrawal were apparent between conditional nulls and control

mice. A hot-plate analgesia meter was also used to activate thermal

nociceptors. This method (Woolfe and MacDonald, 1944) isolates

the responses due to the supraspinal activity. The mean outcome at

four temperatures for four trials at 47.5 T 0.5; 50 T 0.5; 52.5 T 0.5;

and 55 T 0.5-C was plotted (Fig. 3E). Two-way ANOVA analysis

followed by post-hoc Tukey repeated tests showed that the BDNF

conditional null mice were more sensitive to the stimuli than the

control WT group (P < 0.001). Post-hoc tests showed that this

difference was observable at all the temperatures tested (Fig. 3E).

Tonic responses to intraplantar formalin

The biphasic response to intraplantar formalin was observed in

mice as described (Hunskaar et al., 1985). The first phase (0–10

min) showed no significant difference in pain behavior between the

two groups, but the second phase (11–60 min) was reduced

approximately 2.5-fold in the conditional null mutants (Fig. 4A).

Primary hyperalgesia to carrageenan and NGF

Interplantar injection with the inflammatory agent carrageenan

leads to a pronounced thermal hyperalgesia that was maintained

over a period of 24 h (Fig. 4B). The BDNF conditional null

mutants showed a substantial diminution in pain behavior of about

50% at all time points (two-way ANOVA, P = 0.05).

Intraplantar injection of 5 Al of carrier solution containing 50 ngNGF elicited a marked thermal hyperalgesia in the ipsilateral but

not contralateral paw, which persisted until the end of the

experiment 24 h later (Fig. 5A). Earlier findings (Andreev et al.,

1995) demonstrated that the response was biphasic: an acute phase

of hyperalgesia, with rapid onset with some recovery at 1 h p.i.,

followed by a second period of hyperalgesia which appeared by 2

h and was resolved 24 h later. We measured hyperalgesia from 30

min and saw less hyperalgesia in the conditional null mutants at all

time points. There was a highly significant difference in response

between the two groups (two-way ANOVA, P < 0.001; n = 6).

Secondary hyperalgesia to muscle inflammation

Intramuscular injection of 1 Ag NGF into the biceps femoris of

C57/Bl/6/J mice resulted in significantly increased EMG responses

to stimulation of the plantar surface of the foot of the injected leg

with von Frey hair number 18 when compared to the foot of the

uninjected leg (0.29 T 0.02 Vsecs vs. 0.15 T 0.03 Vsecs; t test, P <

0.05). In BDNF conditional null mutants, this effect was not

apparent. The same dose of NGF (1 Ag I.M.) failed to evoke

hyperalgesia in the injected leg when compared to the uninjected

leg (0.15 T 0.01 Vsecs vs. 0.19 T 0.03 Vsecs; t test, P > 0.05) when

stimulated with the same von Frey hair. There were no significant

differences in the baseline (unstimulated) EMG activity between

BDNF conditional null and C/57/Bl/6/J mice (Fig. 5B). A

statistically significant difference in the percentage difference

between injected vs. non-injected legs in the two groups was

observed. EMG activity was 49 T 18% greater in the injected

versus the non-injected leg in wild-types, but, in homozygotes,

activity was unchanged following NGF application with mean

percentage difference between the injected and non-injected legs

being �17 T 15% (Fig. 5C).

Neuropathic pain

The Chung model of spinal nerve ligation was used to assess

the alteration in mechanical sensitivity characteristic of neuropathic

pain in mice (Kim and Chung, 1992). Following a Chung ligature

of BDNF conditional nulls and littermates, the mechanical

allodynia that developed was identical in both sets of animals

(Fig. 6). The time course of development of neuropathic pain was

also identical, with little reversal at 30 days. Although these data

discount BDNF release from Nav1.8-expressing nociceptors as a

critical element in neuropathic pain, they do not rule out a role for

BDNF derived from other peripheral or central sources.

Fig. 2. (A) qPCR was used to compare the expression of BDNF mRNA

with actin in conditional null and wild-type mice. A substantial loss of

BDNF mRNA is apparent. (B) Immunocytochemical staining of BDNF

(red) and NF-200 immunoreactivity (green) showed a qualitative loss of

BDNF-positive cells in the conditional null consistent with the qPCR data.

(C) Cell numbers and proportions of neurofilament and peripherin

expressing neurons were normal in BDNF conditional null animals

compared to floxed littermates. Controls—white symbols, BDNF condi-

tional null—black symbols. (D) Absolute numbers of neurons in L4 DRG

staining for peripherin (black), N52 (gray) and both markers (white) are

shown for wild-type and conditional BDNF mutant mice (KO).

J. Zhao et al. / Mol. Cell. Neurosci. 31 (2006) 539–548 541

Biochemical events associated with inflammatory pain

We used antibodies directed against phosphorylated residues

in glutamate receptors to examine the biochemical correlates of

formalin-induced pain behavior. Formalin induces an increase of

NMDA receptor NR1 (Ser897, Ser896) phosphorylation in the

dorsal spinal cord of control floxed BDNF mice. Western blot

analysis showed that phospho-NR1 (Ser897 and Ser896) could be

detected in the contralateral spinal cord, but there was no

increase in phosphorylation in response to formalin. Formalin

induced a significant increase of phospho-NR1 (Ser897 and

Ser896) in the ipsilateral spinal cord (L3–L5) of control floxed

BDNF mice compared with the contralateral side (Figs. 7A and

B). Values of phospho-NR1 in formalin-stimulated cord com-

pared with control phospho-NR1 were 148 T 13.7% (Ser897; t

test, P < 0.05; n = 4) and 152 T 10.9% (Ser896; t test, P < 0.05;

n = 4). In null mutants, formalin failed to induce any increase of

phospho-NR1 Ser897 or Ser896 in the spinal cord compared with

saline control. Similarly, formalin did not induce GluR1 (Ser831)

phosphorylation in the spinal cord of floxed BDNF control mice

or BDNF conditional null mice (Fig. 7C). Thus, BDNF released

from primary afferents seems to be required for the phosphor-

ylation of NMDA NR1 subunits on Ser896 and 897, while GluR1

Ser831 is not regulated by BDNF released from Nav1.8-positive

primary afferent fibers.

Extracellular-signal-regulated kinase (ERK) is also known to

be activated by inflammatory stimuli such as formalin in a BDNF-

dependent manner (Pezet et al., 2002a,b). Injection of 20

Al formalin induced in floxed BDNF control mice a robust

induction of ERK phosphorylation in the spinal cord ipsilateral to

the injection of formalin. This activation was observed mostly in

superficial laminae (10–17 neurons per section in the superficial

laminae, 5–7 in deeper laminae) in lumbar levels L3 to L5 (Fig.

8). The contralateral site showed only few positive p-ERK

neurons in deep laminae of the cord (3–5 neurons per section).

In BDNF conditional null mice, the induction of ERK was

significantly reduced compared to floxed BDNF control mice in

all L3 to L5 levels (t test, P < 0.05; Fig. 8) in superficial layers of

the cord. In deep laminae of the cord in contrast, where it is

known that BDNF is not abundant, the induction of ERK was not

modified in BDNF conditional null mice, compared to floxed

BDNF controls.

Discussion

Target validation using null mutant mice has proved valuable

in assessing new approaches to analgesic drug development.

When gene deletion causes perinatal lethality, as with BDNF

null mutant mice, tissue-specific gene ablation may provide

important information about the possible relevance of a new

drug target. In addition, identifying the source of release for

neuromodulators such as BDNF provides additional insights into

physiological mechanisms. BDNF heterozygotes are viable and

have provided some insights to BDNF function, although the

absolute levels of BDNF in different tissues have not been

quantitated, while it has proved possible to record electrophys-

iologically from homozygous null mutants up to about day 7.

Fig. 3. (A) Rota-rod studies showed no motor deficits in conditional BDNF null animals, (B) acute mechanical pressure applied with the Randall–Sellito

apparatus also demonstrated identical behavior in nulls and wild-type mice. Responses to low-threshold mechanical stimulation by von Frey hairs are normal

(C) in the null mutant compared to littermate controls. (D) Hargreaves apparatus demonstrates identical latencies of response to thermal stimulation, while

supra-spinal reflexes to heat (E) are sensitized in the null mutant animals. Controls—white symbols, BDNF conditional null—black symbols. ***P < 0.001.

J. Zhao et al. / Mol. Cell. Neurosci. 31 (2006) 539–548542

However, behavioral studies of the role of BDNF in pain

pathways have relied on exogenous application of high doses of

BDNF or the effects of neutralizing receptor bodies that

sequester both NT4 and BDNF. Here, we have investigated

the effects of deleting BDNF expression in Nav1.8-positive,

mainly nociceptive sensory neurons, on pain behavior and

dorsal horn responses to noxious stimuli in an attempt to assess

the role of peripheral BDNF in regulating pain behavior. These

studies are unable to provide information about BDNF

expressed in Nav1.8-negative sensory neurons; the expression

of BDNF is, however, known to be plastic, altering in different

sensory neuron populations in different pain states (Obata et al.,

2003). Nevertheless, where clear pain behavioral deficits occur,

then these can be ascribed to the loss of BDNF in Nav1.8-

positive predominantly nociceptive neurons. The genetic back-

ground of the Cre mouse has been extensively studied and is

unlikely to produce a contribution to altered pain behavior

(Stirling et al., 2005).

Despite the presence of a normal complement of sensory

neurons, there was increased pain responsiveness to noxious

thermal stimulation in the hot-plate test. Mechanical thresholds

were unchanged. In global BDNF heterozygous animals, there is,

in contrast, no change in thermal and mechanical thresholds to

acute pain (MacQueen et al., 2001). The second phase of the

formalin response has, however, been shown to be attenuated in

BDNF heterozygous null mutants (MacQueen et al., 2001).

Results obtained with the conditional sensory neuron BDNF null

mice are consistent with these data. Here, we found that

activation of nociceptors by formalin caused a BDNF release-

dependent increase in NMDA receptor and ERK phosphorylation

that correlates with second phase pain behavior. This mechanism

may also contribute to carrageenan and NGF-induced hyper-

algesia which are both partially reversed in the absence of

predominantly nociceptor-derived BDNF. NGF has been shown

to be involved in nociception and pathological pain conditions

(Lewin et al., 1993; Apfel, 2000). Application of exogenous

NGF has been shown to reduce both thermal and mechanical

withdrawal thresholds (Lewin et al., 1993). Interestingly, thermal

responses at the earliest time points measured (30 min) were

Fig. 4. (A) Intraplantar formalin caused two phases of licking behavior,

the second of which is attenuated in the BDNF conditional null mouse.

(B) Intraplantar injection of carrageenan caused a long-term thermal

hyperalgesia that is attenuated in BDNF conditional nulls. Controls—

white symbols, BDNF conditional null—black symbols. **P < 0.01;

***P < 0.001.

Fig. 5. (A) Intraplantar NGF injections that cause thermal hyperalgesia

resulted in a diminished hyperalgesia in conditional BDNF nulls (open

triangles—saline-injected controls, white squares—NGF-injected control

mice, black squares—NGF-injected conditional BDNF nulls). (B) Sec-

ondary hyperalgesia quantitated electrophysiologically after intra-muscular

injections of NGF were also completely abolished in the conditional

BDNF null mouse. Controls—white symbols, BDNF conditional null—

black symbols. (C) Percentage differences in EMG activity between injected

and non-injected legs are significantly different between groups. Controls—

white symbols, BDNF conditional null—black symbols. *P < 0.05;

**P < 0.01.

J. Zhao et al. / Mol. Cell. Neurosci. 31 (2006) 539–548 543

attenuated in the conditional null, although this behavior has

been considered to reflect peripheral sensitization; our results

suggest a role for BDNF in this process. The actions of NGF are

not limited to effects in the periphery (Bennett, 2001). The

partial reversal of thermal hyperalgesia in the BDNF null

suggests that, while a substantial part of the hyperalgesic action

is mediated by BDNF release in the dorsal horn, other

mechanisms and mediators must also be involved. In contrast,

the mechanical secondary hyperalgesia that NGF evokes can be

partially, if not completely, attributed to extrasynaptic effects of

BDNF.

BDNF is known to play an important role in regulating

synaptic plasticity, and a role in altering pain thresholds has been

proposed both for inflammatory and neuropathic pain. In the

hippocampus, BDNF regulates the expression of the chloride

transporter KCC2 to alter the chloride potential and increase

neuronal excitability (Rivera et al., 2004). A similar mechanism

has been proposed to explain some of the central changes that

occur in neuropathic pain (Coull et al., 2003). However, in the

model of neuropathic pain investigated here, no role for

predominantly nociceptor-derived BDNF could be established.

Yajima et al. have shown that Trk-B receptor bodies can

completely abolish allodynia and thermal hyperalgesia in a

mouse sciatic nerve ligation models of neuropathic pain (Yajima

et al., 2002). Furthermore, they demonstrated that BDNF hetero-

zygotes show attenuated thermal and mechanical hypersensitivity

in the same model. Inhibitors of TRK tyrosine kinase activity and

protein kinase C inhibitors also blocked neuropathic pain. These

and other studies strongly support a role for BDNF in the

development of neuropathic pain. The normal levels of neuropathic

pain in the BDNF conditional null mutants could result from

BDNF released from Nav1.8-negative neurons or BDNF released

from central nervous system neurons. It is also known that both

support cells and immune cells produce neurotrophins, and these

are also a potential source of the BDNF that may contribute to

neuropathic pain.

In summary, these studies confirm and extend the case of a

highly significant role for nociceptor-derived BDNF rather than

NT4 in mediating inflammatory pain caused by NGF, carrageenan

and formalin. If BDNF in the spinal cord does play a role in

neuropathic pain, as seems likely, it must be derived from a distinct

Fig. 6. Sciatic nerve ligation following the Chung procedure caused a long-

lasting mechanical hyperalgesia, although thermal thresholds were unaf-

fected (not shown). Both floxed BDNF and conditional deleted BDNF

showed an identical time course and level of allodynia after this injury.

Controls—white symbols, BDNF conditional null—black symbols.

Fig. 7. (A) Formalin increases NR1 (Ser897) phosphorylation in the spinal

cord of floxed BDNF mice but not in BDNF conditional null mutants.

Proteins were extracted from spinal cords (L3–L5) 30 min after formalin

injection. The top blot shows the immuno-reactive bands detected with anti-

phospho-NR1 antibody. The bottom blot shows GAPDH immunoreactivity

to compare the amount of proteins in each lane in the same membrane. The

relative phospho-NR1 (Ser897) protein levels are expressed as a percentage

of the vehicle control. (B) Formalin also increases NR1 (Ser896)

phosphorylation. The relative phosphor-NR1 (Ser896) protein levels are

expressed as a percentage of the vehicle control. (C) No alteration in AMPA

GluR1 receptor phosphorylation on Ser831 was evoked by formalin in either

floxed or BDNF conditional null mutants. *P < 0.05; **P < 0.01.

J. Zhao et al. / Mol. Cell. Neurosci. 31 (2006) 539–548544

cellular source than Nav1.8-positive sensory neurons. These studies

support a therapeutic approach to inflammatory pain based on

down-regulating peripheral levels of BDNF in sensory neurons.

Experimental methods

Generation of Nav1.8-specific BDNF knock-out mice

Floxed mice containing loxP sites flanking BDNF exon 5 (Rios et

al., 2001) were crossed with the Nav1.8-Cre strain (Stirling et al., 2005)

to affect BDNF gene ablation in a defined subset of sensory neurons.

The study population contained the homozygous floxed BDNF gene and

one copy of the Nav1.8-Cre allele, while homozygous floxed BDNF

littermates were used as controls. Genotyping of all animals was done

with PCR as previously described (Stirling et al., 2005; Rios et al.,

2001). Mice were housed with a 12-h light:12-h (lights on at 07:00)

dark cycle and maintained under standard condition (21 T 1-C, food and

water ad libitum). Behavioral studies were carried out during the light

cycle. Experiments were carried out on female animals between 2 and 3

months old, drawn from different litters to avoid any ‘‘litter effect’’ and

tested blind. Real-time RT-PCR analysis of BDNF was performed using

an iCycler (Bio-Rad, CA). DRG RNA was extracted using TRIzolR

reagent (Gibco BRL), treated with RQ1 RNase-free DNase (Promega)

and equal amounts of total RNA were reversed transcribed using random

hexamers, and Superscripti II RT (Invitrogen) PCR reactions were

performed using PlatinumR SYBRR Green qPCR Supermix UDG

(Invitrogen) and BDNF gene-specific primers, forward primer: 5V-GCATCTGTTGGGGAGACAAG-3V; reversed primer: 5V-TGGTCAT-CACTCTTCTC ACCTG-3V. Reactions were performed in triplicate,

and threshold cycle values were normalized to h-Actin gene expression,

forward primer: 5V-TCTGTGTGGATCGGTGGCTC-3V; reversed primer:

5V-CTGCTTGCTGATCCACAT-CTG-3V. The specificity of the products

was determined by melting curve analysis, and their correct sizes were

analyzed by electrophoresis. The ratio of the relative concentration of

BDNF to h-Actin of each sample was calculated by using the 2DCT

formula.

Cell counts and immunocytochemistry

DRG 11 AM sections were blocked in 10% goats’ serum in PBS

overnight at 4-C then incubated in a mixture of 1:1000 NF200 antibodies

(mouse monoclonal, Sigma) and 1:1000 anti-peripherin antibody (rabbit

polyclonal, Chemicon) diluted in 10% goat serum in PBS overnight at 4-C.The slides were then washed in three changes of PBS (2 � 5 min, 1 � 30

min). They were then incubated with 1:600 FITC-conjugated goat anti-

Fig. 8. Activation of the MAPK ERK induced by intraplantar formalin is reduced in BDNF conditional null mice. (A–D) Representative immunostaining for p-

ERK in floxed control mice (A–B) or BDNF KO (C–D) 5 min after the injection of formalin in superficial (laminae I, II of Rexed, graph on the left) or (F) in

deep layers of the cord (laminae III–VI). The right hemicord on the picture is the cord ipsilateral to the injection of formalin. B and D are higher power

magnification of A and C, respectively. (E) Quantification of the mean number (TSEM) of positive p-ERK neurons in the spinal cord L3 to L5 ipsilateral to the

injection of formalin in superficial (graph on the left) or (F) in deep layers of the cord. Scale bar for figures A–D; A, C: 94 Hm; B, D: 34 Hm. *P < 0.05.

J. Zhao et al. / Mol. Cell. Neurosci. 31 (2006) 539–548 545

mouse IgG and 1:1000 Alexa-Fluor-conjugated goat anti-rabbit IgG for 3

h at room temperature. The sections underwent three more washes in PBS

(2 � 5 min, 1� overnight) and were then cover-slipped with PBS/Glycerol

(CITIFluor).

The number of neurons was counted in every 8th section throughout the

DRG. Thus, the DRG was sampled every 88 Al to avoid double counting of

the large diameter cells. Every visible cell was counted whether the

nucleolus was present or not. Each DRG was counted twice, and the results

were pooled. The percentages of NF200-positive, peripherin-positive and

double-stained cells were calculated for each section. Mean and SE of these

percentages were evaluated for wild-type and mutant groups. The raw data

from the cell counts passed the test for normality (Kolmogorov–Smirnov,

P > 0.200 for all groups), so significance was determined using a two-tailed

unpaired heteroscedastic t test.

Other primary antibodies used: monoclonal mouse anti-neurofilament-

H antibody, N52 (1:1000, Sigma), anti-BDNF rabbit antiserum (Abcom

ab6201, 1:400). The following secondary antibodies were used: anti-mouse

(goat) (Fab)2 FITC (1:1000, Jackson) and anti-rabbit Alexa Fluor 594 goat

IgG (1:1000, Molecular Laboratories).

Western immunoblot analysis

After 30 min of treatment with formalin (20 Al of 5% formalin plantar

surface injection in right paw, 20 Al of saline in left paw), floxed BDNF

mice and BDNF conditional null mice were killed. The dorsal half of the

L3–L5 spinal cord tissues was removed and homogenized (50 mM Tris–

Cl pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% NP40, 0.5% Na-

deoxycholate, 0.1% SDS, 1 mM Na3VO4, 5 mM NaF, 1 mM PMSF, 1

U/ml aprotinin, 10 Ag/ml antipain, 1 Ag/ml leupeptin and 1 Ag/ml

pepstatin A). The homogenate was centrifuged at 14,000 rpm for 20 min

at 4-C. The supernatant was removed. The protein concentration was

determined with a BCA Kit. Proteins (40 Ag) were separated on a 7.5%

SDS-PAGE gel and blotted to nitrocellulose membrane with a Bio-Rad

Transfer Cell system. The blots were blocked with blocking buffer 1 (TBS

contains 0.1% Triton X-100 and 0.1% BSA) at room temperature for 1 h.

The blots were washed with TBST (TBS containing 0.1% Tween20) for

anti-phospho-NR1 Ser897 twice or PBST (PBS containing 0.1% Tween20)

for anti-phospho-NR1 Ser896 twice. After a rinse in water, the membrane

was incubated again with TBST–MLK (TBST containing 5% nonfat dry

milk, for anti-phospho-NR1 Ser897) or PBST–MLK (PBST containing 5%

nonfat dry milk, for anti-phospho-NR1 Ser896 and anti-phospho-GluR1

Ser831) at room temperature for 1 h. Blots were incubated in blocking

buffer 2 with the respective antibody overnight at 4-C. The membrane

was washed with TBST or PBST and incubated for 1 h with anti-goat IgG

horseradish peroxidase (1:2000) in TBST–MIL or PBST–MLK at room

temperature for 1 h. The membrane was then washed three times with

TBST or PBST, and immunoreactivity was finally detected by using ECL

kits (Amersham). Antibodies were used at dilutions suggested by the

makers instructions: anti-phospho-NR1 Ser897 (Upstate), anti-phospho-

NR1 Ser896 (Upstate), anti-phosphor-GluR1 Ser831 (Upstate), anti-

GAPDH (Chemicon), anti-rabbit IgG horseradish peroxidase (Amersham),

anti-mouse IgG horseradish peroxidase (Amersham).

Immunohistochemistry for p-ERK

Adult floxed BDNF mice (n = 4) or BDNF conditional mice (n = 4)

were anaesthetized with urethane (1.25 g/kg, i.p.) and received intraplantar

injections of formalin (Sigma, 20 Al injected in plantar surface of the left

hind paw). Five minutes after the injection, animals were transcardially

perfused with 30 ml heparinized saline (0.9% w/v NaCl) followed by 100

ml of 4% w/v paraformaldehyde in 0.1 M phosphate buffer pH 7.4 (PFA),

15% of a saturated solution of picric acid. The spinal cord was post-fixed in

the same fixative overnight and cryoprotected overnight in 20% w/v

sucrose in 0.1 MPB at 4-C. The spinal cords were embedded in OCT

embedding compound (BDH) on liquid nitrogen and cut serially (20 Amthickness) on a cryostat and collected onto superfrost slides (BDH). Every 6

sections were kept for p-ERK immunostaining. After several washes in

PBS, slides were incubated overnight in mouse anti-phosphorylated-ERK1/

ERK2 (recognizing sites Thr 202 and Tyr 204, New England Biolabs, UK,

1:200). After several washes in PBS, sections were incubated in goat anti-

rabbit Alexa Fluor 488 antibody (Molecular Probes, 1:1000) for 2 h.

Finally, slides were washed and mounted with Vectashield medium (Vector

laboratories). p-ERK staining was visualized using a fluorescent Leica

microscope. Images were taken using Hamamatsu Camera and software.

The same setup of acquisition was used to acquire the pictures of both

groups of mice. In each animal, the number of positive p-ERK neurons was

counted blind in 5 sections of the following lumbar spinal cord levels: L3,

L4 and L5.

Electrophysiology

Mice were injected with 1 Ag NGF unilaterally in the biceps femoris 24

h prior to EMG recording. Animals were subsequently anesthetized with

isoflurane (2%) and ventilated. Animals were mounted in a small animal

spinal frame and hindlimbs secured in a slight extension with the plantar

foot surface exposed for cutaneous stimulation. Bipolar EMG electrodes

(Ainsworks, London) were placed through a small incision into the belly of

the biceps femoris. Raw signals were amplified using a headstage amplifier

(NL100, Neurolog, Digitimer), preamplified and filtered (NL104, NL125)

and displayed on a digital storage oscilloscope (Hameg HM205). The

signal was fed to an analogue-to-digital signal converter for further analysis

using MacLab software (PowerLab 4S, AD Instruments, Castle Hill,

Australia). von Frey hairs (Stoelting U.S.A.) of graded intensity were

applied to the hindpaw and the EMG response recorded. The mechanical

withdrawal threshold was defined as the lowest number von Frey hair that

elicited an EMG response. Up to three vFh above threshold were

sequentially applied at 1-min intervals. Recordings were made from both

limbs, and the order in which this was done was randomized. Animals were

equilibrated for 30 min on the frame prior to recordings. The percentage

difference in EMG activity between injected and non-injected legs was

calculated, and comparisons between homozygote and wild-type mice

performed.

Behavioral analysis

Rota-rod test

Mice were acclimatized to the stationary rod (model 7650, Ugo Basile,

Italy) for 120 s (Crawley and Paylor, 1997), and 5 s later, the rod was

rotated at a constant speed of 20 rpm for 300 s. The time taken for the

animal to either fall off or rotate passively was recorded. Three further trials

(one trial per day) with the rod accelerating from 20 rpm to the maximum of

40 rpm within the 5-min test period were carried out. The time that each

mouse was able to stay on the rod without either falling or passively

rotating was recorded. The mean T SEM was determined and analyzed with

a Student’s t test.

Randall–Sellito test

Mice were passively restrained in a Perspex tube allowing the apparatus

to access the tail. After a 15-min acclimatization period, the area of the tail

approximately 1 cm from the base was subjected to an increasing pressure

and the force (grams) at which a struggling response was elicited noted.

This was repeated three times, and the mean T SEM calculated and assessed

by means of a Student’s t test (Randall and Sellito, 1957).

Formalin test

The two groups of animals (n = 6 each group) were singly housed in

Perspex boxes and given 30 min to habituate to the testing environment.

Animals were then injected subcutaneously with formalin (15 Al, 5%

dilution of stock formalin (40% w/v) in saline). Nociceptive behavior was

taken to be licking and biting the injected paw only. The time that these two

activities were displayed by the animal was recorded in 5-min bins until 60

min had passed. Differences between the two groups were assessed with

two-way ANOVA followed by post-hoc Tukey tests (McCall et al., 1996;

Hunskaar et al., 1985).

J. Zhao et al. / Mol. Cell. Neurosci. 31 (2006) 539–548546

Hot-plate test

The response to thermal stimuli was tested using a hot-plate

analgesia meter (Ugo Basile, Italy) (Crawley and Paylor, 1997). Animals

were habituated for the equipment for 15 min before the basal metal

plate was heated to the test temperature. The time taken for a response

was recorded. Lifting of any paw, biting and licking, rearing up on hind

legs, paw flinching or jumping was taken as the cut-off point with a

maximum of 60 s for temperatures under 52.5 T 0.5-C and 30 s above

53 T 0.5-C. One test was conducted per animal per day. Four

temperatures were assessed on subsequent days with each test group

having 12 animals. The means for each temperature T SEM were

determined and subjected to a two-way ANOVA test of significance

followed by post-hoc Tukey test.

von Frey test of mechanical thresholds

After acclimatization to the testing environment (30 min), mechan-

ical sensory thresholds were determined by paw withdrawal to

application of a series of von Frey filaments to the glabrous surface

of the hind paws. Calibrated von Frey filaments were applied five times

per paw with enough force to cause buckling of the filament. Eight

filaments were tested in ascending order from 0.219 g to 7.59 g. The

percentage response for each filament was determined by scoring the

positive responses (both supraspinal; biting, licking and simple hyper-

reflexia; paw lifting); i.e. number of trials accompanied by a response

divided by 5 and multiplied by 100. Left and right hind paws were

tested with 3-min breaks between subsequent tests. Each group

contained 12 animals, and the mean T SEM was assessed using two-

way ANOVA with post-hoc Tukey tests.

Hargreaves test of thermal nociceptive thresholds

The Hargreaves method (Hargreaves et al., 1988) was used to measure

thermal hyperalgesia using the plantar test (Hargreaves’ Basile Plantar test

model 7370; Ugo Basile, Comerio, Italy). Mice were habituated for 30 min

to the apparatus. The equipment was calibrated to give a mean paw

withdrawal latencies (PWL) of approximately 10 s by adjusting the infra-

red (IR) intensity. PWLs were taken three times for both hind paws with at

least 5-min intervals between each subsequent test. The mean of the three

measures represented the latency of paw withdrawal and was taken as a

measure of thermal pain responses. A cut-off point of 20 s was used to

prevent tissue damage.

Carrageenan and nerve growth factor (NGF) induced thermal hyperalgesia

Mice were injected either with 20 Al carrageenan (2%, w/v) or 50 ng

NGF (5 Al carrier volume) (human recombinant nerve growth factor 4.2

mg/ml in 20 mM succinate buffer diluted to 10 Ag/ml with saline, Sigma

Chemicals, UK) into the subcutaneous plantar surface of the left hind paw.

The injection was done under anesthesia induced by 4% halothane/oxygen

to ensure reproducible injections of a consistent position and depth. The

animals recovered within 3 min. The development of the thermal

hyperalgesia was tracked using the Hargreaves apparatus after the injection

up to a maximum of 24 h. Two-way ANOVA was carried out followed by

post-hoc Tukey analysis.

Sciatic nerve injury

Baseline thermal and mechanical thresholds were recorded from null

and wild-type littermates using Hargreaves test and von Frey hairs (using

the up–down method (Chaplan et al., 1994)) respectively. Animals were

anesthetized using halothane. A midline incision was made in the skin of

the back at the L4–S2 levels and the left paraspinal muscles separated from

the spinous processes, facet joints and transverse processes at the L4–S1

levels. The left L5 spinal nerve was tightly ligated using silk thread (Kim

and Chung, 1992). Mechanical thresholds were measured after injury, and

the results for the two groups were compared using a two-way repeated

measures analysis of variance test. Results of the 50% withdrawal threshold

were expressed as the relative change in the 50% threshold at each time

point after injection (test/baseline) for each group T SEM. The results for

the two groups were compared using a two-way repeated measures analysis

of variance test.

Statistical analysis

All data are presented as mean T SEM. Data were assessed for

normality, and normally distributed data sets were compared with two-way

analysis of variance (ANOVA) followed by post-hoc Tukey multiple

comparison tests. Non-normal data were assessed with Student’s unpaired t

test. P < 0.05 was regarded as significant. All calculations were done using

SigmaStat 2.01.

Acknowledgments

We acknowledge Sarah E. Slack for great help in detection of

phosphorylation of NMDA receptors in spinal cord. We also thank

the MRC, the BBSRC and the Wellcome Trust for funding this

work.

References

Andreev, N.Y., Dimitrieva, N., Koltzenburg, M., McMahon, S.B., 1995.

Peripheral administration of nerve growth factor in the adult rat

produces a thermal hyperalgesia that requires the presence of

sympathetic post-ganglionic neurones. Pain 63, 109–115.

Apfel, S.C., 2000. Neurotrophic factors and pain. Clin. J. Pain 16, S7–S11.

Bennett, D.L., 2001. Neurotrophic factors: important regulators of

nociceptive function. Neuroscientist 7, 13–17.

Brenner, G.J., Ji, R.R., Shaffer, S., Woolf, C.J., 2004. Peripheral noxious

stimulation induces phosphorylation of the NMDA receptor NR1

subunit at the PKC-dependent site, serine-896, in spinal cord dorsal

horn neurons. Eur. J. Neurosci. 20, 375–384.

Chaplan, S.R., Bach, F.W., Pogrel, J.W., Chung, J.M., Yaksh, T.L., 1994.

Quantitative assessment of tactile allodynia in the rat paw. J. Neurosci.

Methods 53, 55–63.

Cho, H.J., Kim, J.K., Zhou, X.F., Rush, R.A., 1997a. Increased brain-

derived neurotrophic factor immunoreactivity in rat dorsal root ganglia

and spinal cord following peripheral inflammation. Brain Res. 764,

269–272.

Cho, H.J., Kim, S.Y., Park, M.J., Kim, D.S., Kim, J.K., Chu, M.Y., 1997b.

Expression of mRNA for brain-derived neurotrophic factor in the

dorsal root ganglion following peripheral inflammation. Brain Res.

749, 358–362.

Cho, H.J., Kim, J.K., Park, H.C., Kim, J.K., Kim, D.S., Ha, S.O., Hong,

H.S., 1998. Changes in brain-derived neurotrophic factor immunoreac-

tivity in rat dorsal root ganglia, spinal cord, and gracile nuclei following

cut or crush injuries. Exp. Neurol. 154, 224–230.

Coull, J.A., Boudreau, D., Bachand, K., Prescott, S.A., Nault, F., Sik, A.,

De Koninck, P., De Koninck, Y., 2003. Trans-synaptic shift in anion

gradient in spinal lamina I neurons as a mechanism of neuropathic pain.

Nature 424, 938–942.

Crawley, J.N., Paylor, R., 1997. A proposed test battery and constellations

of specific behavioral paradigms to investigate the behavioral pheno-

types of transgenic and knockout mice. Horm. Behav. 31, 197–211.

Eaton, M.J., Blits, B., Ruitenberg, M.J., Verhaagen, J., Oudega, M., 2002.

Amelioration of chronic neuropathic pain after partial nerve injury by

adeno-associated viral (AAV) vector-mediated over-expression of

BDNF in the rat spinal cord. Gene Ther. 9, 1387–1395.

Ernfors, P., Wetmore, C., Olson, L., Persson, H., 1990. Identification of

cells in rat brain and peripheral tissues expressing mRNA for members

of the nerve growth factor family. Neuron 5, 511–526.

Fukuoka, T., Kondo, E., Dai, Y., Hashimoto, N., Noguchi, K., 2001. Brain-

derived neurotrophic factor increases in the uninjured dorsal root

ganglion neurons in selective spinal nerve ligation model. J. Neurosci.

21, 4891–4900.

J. Zhao et al. / Mol. Cell. Neurosci. 31 (2006) 539–548 547

Garraway, S.M., Petruska, J.C., Mendell, L.M., 2003. BDNF sensitizes the

response of lamina II neurons to high threshold primary afferent inputs.

Eur. J. Neurosci. 18, 2467–2476.

Garraway, S.M., Anderson, A.J., Mendell, L.M., 2005. BDNF-induced

facilitation of afferent evoked responses in lamina II neurons is reduced

following neonatal spinal cord contusion injury. J. Neurophysiol. 94,

1798–1804.

Guo, W., Zou, S., Guan, Y., Ikeda, T., Tal, M., Dubner, R., Ren, K., 2002.

Tyrosine phosphorylation of the NR2B subunit of the NMDA receptor

in the spinal cord during the development and maintenance of

inflammatory hyperalgesia. J. Neurosci. 22, 6208–6217.

Ha, S.O., Kim, J.K., Hong, H.S., Kim, D.S., Cho, H.J., 2001. Expression of

brain-derived neurotrophic factor in rat dorsal root ganglia, spinal cord

and gracile nuclei in experimental models of neuropathic pain.

Neuroscience 107, 301–309.

Hargreaves, K., Dubner, R., Brown, F., Flores, C., Joris, J., 1988. A new

and sensitive method for measuring thermal nociception in cutaneous

hyperalgesia. Pain 32, 77–88.

Heppenstall, P.A., Lewin, G.R., 2001. BDNF but not NT-4 is required

for normal flexion reflex plasticity and function. Proc. Natl. Acad. Sci.

U. S. A. 98, 8107–8112.

Huang, E.J., Reichardt, L.F., 2003. Trk receptors: roles in neuronal signal

transduction. Annu. Rev. Biochem. 72, 609–642.

Hunskaar, S., Fasmer, O.B., Hole, K., 1985. Formalin test in mice, a

useful technique for evaluating mild analgesics. J. Neurosci. Methods

14, 69–76.

Jones, K.R., Farinas, I., Backus, C., Reichardt, L.F., 1994. Targeted

disruption of the BDNF gene perturbs brain and sensory neuron

development but not motor neuron development. Cell 76, 989–999.

Kerr, B.J., Bradbury, E.J., Bennett, D.L., Trivedi, P.M., Dassan, P., French,

J., Shelton, D.B., McMahon, S.B., Thompson, S.W., 1999. Brain-

derived neurotrophic factor modulates nociceptive sensory inputs and

NMDA-evoked responses in the rat spinal cord. J. Neurosci. 19,

5138–5148.

Kim, S.H., Chung, J.M., 1992. An experimental model for peripheral

neuropathy produced by segmental spinal nerve ligation in the rat. Pain

50, 355–363.

Lewin, G.R., Ritter, A.M., Mendell, L.M., 1993. Nerve growth factor-

induced hyperalgesia in the neonatal and adult rat. J. Neurosci. 13,

2136–2148.

Li, Y., Jia, Y.C., Cui, K., Li, N., Zheng, Z.Y., Wang, Y.Z., Yuan, X.B., 2005.

Essential role of TRPC channels in the guidance of nerve growth cones

by brain-derived neurotrophic factor. Nature 434, 894–898.

Liu, X., Ernfors, P., Wu, H., Jaenisch, R., 1995. Sensory but not motor

neuron deficits in mice lacking NT4 and BDNF. Nature 375, 238–241.

MacQueen, G.M., Ramakrishnan, K., Croll, S.D., Siuciak, J.A., Yu, G.,

Young, L.T., Fahnestock, M., 2001. Performance of heterozygous

brain-derived neurotrophic factor knockout mice on behavioral

analogues of anxiety, nociception, and depression. Behav. Neurosci.

15, 1145–1153.

Malcangio, M., Lessmann, V., 2003. A common thread for pain and

memory synapses? Brain-derived neurotrophic factor and trkB recep-

tors. Trends Pharmacol. Sci. 24, 116–121.

McAllister, A.K., Katz, L.C., Lo, D.C., 1999. Neurotrophins and synaptic

plasticity. Annu. Rev. Neurosci. 22, 295–318.

McCall, W.D., Tanner, K.D., Levine, J.D., 1996. Formalin induces biphasic

activity in C-fibers in the rat. Neurosci. Lett. 208, 45–48.

Michael, G.J., Averill, S., Nitkunan, A., Rattray, M., Bennett, D.L., Yan, Q.,

Priestley, J.V., 1997. Nerve growth factor treatment increases brain-

derived neurotrophic factor selectively in TrkA-expressing dorsal root

ganglion cells and in their central terminations within the spinal cord.

J. Neurosci. 17, 8476–8490.

Obata, K., Yamanaka, H., Dai, Y., Tachibana, T., Fukuoka, T.,

Tokunaga, A., Yoshikawa, H., Noguchi, K., 2003. Differential

activation of extracellular signal-regulated protein kinase in primary

afferent neurons regulates brain-derived neurotrophic factor expres-

sion after peripheral inflammation and nerve injury. J. Neurosci. 23,

4117–4126.

Pezet, S., Malcangio, M., Lever, I.J., Perkinton, M.S., Thompson, S.W.,

Williams, R.J., McMahon, S.B., 2002a. Noxious stimulation induces

Trk receptor and downstream ERK phosphorylation in spinal dorsal

horn. Mol. Cell. Neurosci. 21, 684–695.

Pezet, S., Malcangio, M., McMahon, S.B., 2002b. BDNF: a neuromodulator

in nociceptive pathways? Brain Res. Brain Res. Rev. 40, 240–249.

Randall, L.O., Sellito, J.J., 1957. A method for measurement of

analgesic activity on inflamed tissue. Arch. Int. Pharmacodyn. Ther.

111, 409–419.

Rios, M., Fan, G., Fekete, C., Kelly, J., Bates, B., Kuehn, R., Lechan, R.M.,

Jaenisch, R., 2001. Conditional deletion of brain-derived neurotrophic

factor in the postnatal brain leads to obesity and hyperactivity. Mol.

Endocrinol. 15, 1748–1757.

Rivera, C., Voipio, J., Thomas-Crusells, J., Li, H., Emri, Z., Sipila, S.,

Payne, J.A., Minichiello, L., Saarma, M., Kaila, K., 2004. Mechanism

of activity-dependent downregulation of the neuron-specific K–Cl

cotransporter KCC2. J. Neurosci. 24, 4683–4691.

Slack, S.E., Thompson, S.W., 2002. Brain-derived neurotrophic factor

induces NMDA receptor 1 phosphorylation in rat spinal cord. Neuro-

Report 13, 1967–1970.

Slack, S.E., Pezet, S., McMahon, S.B., Thompson, S.W., Malcangio, M.,

2004. Brain-derived neurotrophic factor induces NMDA receptor

subunit one phosphorylation via ERK and PKC in the rat spinal cord.

Eur. J. Neurosci. 20, 1769–1778.

Stirling, L.C., Forlani, G., Baker, M.D., Wood, J.N., Matthews, E.A.,

Dickenson, A.H., Nassar, M.A., 2005. Nociceptor-specific gene

deletion using heterozygous Na(V)1.8-Cre recombinase mice. Pain

113, 27–36.

Thompson, S.W., Bennett, D.L., Kerr, B.J., Bradbury, E.J., McMahon, S.B.,

1999. Brain-derived neurotrophic factor is an endogenous modulator of

nociceptive responses in the spinal cord. Proc. Natl. Acad. Sci. U. S. A.

96, 7714–7718.

Woolfe, G., MacDonald, A.D., 1944. The evaluation of the analgesic action

of pethidine hydrochloride (Demerol). J. Pharmacol. Exp. Ther. 80,

300–307.

Yajima, Y., Narita, M., Narita, M., Matsumoto, N., Suzuki, T., 2002.

Involvement of a spinal brain-derived neurotrophic factor/full-length

TrkB pathway in the development of nerve injury-induced thermal

hyperalgesia in mice. Brain Res. 958, 338–346.

Zhang, J.Y., Luo, X.G., Xian, C.J., Liu, Z.H., Zhou, X.F., 2000.

Endogenous BDNF is required for myelination and regenera-

tion of injured sciatic nerve in rodents. Eur. J. Neurosci. 12,

4171–4180.

Zhou, X.F., Deng, Y.S., Xian, C.J., Zhong, J.H., 2000. Neurotrophins from

dorsal root ganglia trigger allodynia after spinal nerve injury in rats. Eur.

J. Neurosci. 12, 100–105.

J. Zhao et al. / Mol. Cell. Neurosci. 31 (2006) 539–548548