REVIEW ARTICLES

ACTA ANAESTHESIOL SIN 41:187-196, 2003

Implications of Intrathecal Pertussis Toxin Animal Model on

the Cellular Mechanisms of Neuropathic Pain Syndrome

Zhi-Hong Wen, Yi-Chen Chang, Chih-Shung Wong

Department of Anesthesiology, Tri-Service General Hospital and National Defense
Medical Center, Taipei, Taiwan, R.O.C.

Like opioid tolerance, neuropathic pain syndrome manifested by hyperalgesia and allodynia responds poorly to
opioids. Hitherto, its development is still not clear and its treatment and prevention are still disputable. Pertussis
toxin (PTX) which ADP-ribosylates the -subunit of inhibitory guanine nucleotide binding regulatory proteins (Gi/
Go), is used to induce morphine tolerance through intrathecal (i.t.) injection. It decreases the antinociceptive effect
of opioid receptor agonists, and produces a thermal hyperalgesia as well. With treatment of PTX the inhibitory
Gi- and Go-proteins signal transduction is inactivated. Inhibition of the inhibitory system would likely lead to a
predominance of the excitatory system. Intrathecal PTX administration has also been suggested as a model for
study of the central mechanisms of neuropathic pain. In our previous studies, with intrathecal microdialysis and
drug delivery techniques, we correlated the biochemical and pharmacological effects on the behavioral expressions
of i.t. PTX-treated rats. Intrathecal PTX administration would induce thermal hyperalgesia in rats, with accom-
paniments of a prolonged increase in the concentrations of excitatory amino acids (EAAs), glutamate and aspar-
tate, and a decrease in the concentration of the inhibitory amino acid (IAA) glycine in the spinal CSF dialysates.
The PTX-induced thermal hyperalgesia peaked between day 2 and 4, but no cold allodynia is observed; i.t. ad-
ministration of N-methyl-D-aspartate (NMDA) receptor antagonist, D-2-amino-5-phosponovaleric acid (D-AP5),
glycine and protein kinase C (PKC) inhibitor chelerythrine attenuated the thermal hyperalgesia. The PKC content
of both synaptosomal and cytosolic fractions were significantly increased in PTX-treated rats. In contrast, the levels
of PKC , I, or II isozymes in these fractions were unaffected. Infusion of NMDA antagonist D-AP5 prevented
both the thermal hyperalgesia and the increase in PKC expression in PTX-treated rats. Similar to our previous
report, i.t. PTX reduced morphine's analgesic effect. PKC inhibitor chelerythrine attenuated this reduction of mor-
phine's analgesia, and an inhibition of the morphine-evoked EAAs release was observed in PTX-treated rats as well.
Taken together, i.t. PTX-induced neuropathic pain syndrome is accompanied by increasing of EAAs, decreasing of
IAA release, and a selective increasing of PKC expression in the spinal cord. Inhibition of PKC not only blocked
thermal hyperalgesia, but also reversed the reduction of morphine's analgesic effect in PTX-rats. These results sug-
gest that PTX-induced neuropathic pain syndromes are involved in EAAs, IAAs and PKC alternations.

Key words : Pain. Pertussis toxin. Excitatory amino acids. Protein kinase C.

symptoms include hyperalgesia and allodynia. 1,2 Hyper-

N
europathic pain syndrome, usually associated
with central or peripheral nerve injury, responds
poorly to opioids and other analgesics; the major
Received: July 24, 2003.
Revised version received: September 12, 2003.
Accepted for publication: September 15, 2003.
Address correspondence and reprint requests: Dr. Chih-Shung Wong,
Department of Anesthesiology, Tri-Service General Hospital and Na-
tional Defense Medical Center, #325, Chenggung Road, Section 2, Nei-
hu 114, Taipei, Taiwan, R.O.C.
Tel: +886-2-87927128, Fax: +886-2-87927127
E-mail: w82556@ndmctsgh.edu.tw
algesia is characterized by an increasing of pain response
to a suprathreshold noxious stimuli, which results from
abnormal processing of nociceptive inputs. Allodynia is
a sensation of pain elicited by non-noxious stimuli which
can be produced in two ways: either by the action of low
threshold myelinated A fibers on the altered central
nervous system, or by a reduction of the threshold of
nociceptive terminals in the periphery.3 Pertussis toxin
(PTX)-sensitive Gi-protein coupling had been demon-
strated to be involved in the signal transduction of opioid,

187
188 NEUORPATHIC PAIN AND PERUTSSIS TOXIN
2, GABAB , A1-adenosine receptors;4-6 and they are re-
ported to be involved in analgesia (antinociception). 7
Intrathecal injection of PTX not only attenuated opioid's
antinociceptive efficacy, 8-11 but also produced hyperal-
gesia and allodynia.12,13 It has been suggested to be used
as a neuropathic pain model to study the central mechan-
isms of neuropathic pain syndrome. 12,14 In contrast to the
peripheral animal models, such as sciatic nerve ligation
and spinal nerves transection, central nociceptor sensiti-
zation resulted from the peripheral treatments.15,16
The possible mechanisms of reduction of opioids'
antinociceptive effect are (1) down-regulation of func-
tional c-opioid receptors (the high affinity state), (2) re-
duction of total -opioid receptor number, and (3) altera-
tion of intracellular nociception signal transduction, such
as increasing release of EAAs and activation of PKC.
Pervious studies demonstrated that PTX treatment did
not affect total -opioid receptor density and the content
of Gs-, Gi- and Go-proteins on the neuronal plasma mem-
brane from the brain and spinal cord of rats.9,17,18 Chen
et al. demonstrated that the GTP S binding of -opioid
receptor was significantly reduced in the spinal dorsal
horn of diabetic neuropathic pain rats.19 Wong et al. also
found that PTX injection decreased the antinociceptive
effect of -opioid peptide PL017, in association with a
reduction of -opioid receptor high-affinity sites.9 This
impairment of -opioid receptors function may be one of
the mechanisms responsible for the reduction of -opioid
agonists' analgesic effect in neuropathic pain animals.
Intrathecal PTX A New Model for Neuropathic
Pain Study
PTX was first introduced to research on receptor-
coupled signaling by Katada and Ui. 20,21 PTX is a hexa-
mer (117,000 KDa) of five dissimilar subunits that were
named in order of decreasing molecular weight (S1 -S5 ).22
The S1 -subunit exhibits enzymatic activity of ADP-ribo-
syltransferase, which enters the cell together with the
bound glycoproteins by internalization with the help of
S2 -S5 subunits. 23,24 PTX has been known to inactivate
Gi/Go-proteins by ADP-ribosylation of the specific cys-
tein on C-terminal, by disrupting the inhibitory signal
transduction.4,25 Gi/Go-proteins regulate many intracel-
lular signal transduction and channel activity, such as
inhibiting adenylate cyclase activity, opening potassium
channel, closing calcium channels, and breaking down
inositol phospholipids. 26-29 With heterotrimeric structure
all of the G-proteins acting as membrane signal transdu-
cers consist of nucleotide binding -, - and -subunits.
There are at least 16 types of -subunit, along with at
least seven and five isoforms. Activation of G-protein
coupled receptors leads to the dissociation of G-protein
into - and -subunits. Both - and -subunits are able
to activate G-protein-linked second messenger system.30
For example, -opioid receptor agonists would elicit bio-
logical actions through decrease of production of cAMP,
opening Gi protein-mediated inwardly rectifying potas-
sium channels, and closing of voltage-dependent calcium
channels.31,32 PTX increases the release of excitatory
amino acid (EAA) glutamate from chromaffin cells, cere-
bellar granule neurons, and dorsal root ganglion cells
by modulating Ca2+ channel activity. 33-35 Durcan and
Morgan also reported that PTX enhanced N-methyl-
D-aspartate (NMDA)-induced seizures in mice.36 When
i.t.-PTX-treated rats were used as a morphine tolerance
model, we observed both a reduction of opioids' analge-
sic effect and thermal hyperalgesia. 9 A reduction of
-opioid receptor high-affinity sites was also observed
in the same study.9 NMDA receptor antagonists not
only prevented the development of morphine tolerance
but also inhibited the PTX-induced neuropathic pain syn-
drome.9,12 Activation of NMDA and down stream PKC
phosphorylation are crucial in the development of neu-
ropathic pain. Activation of PKC could down regulate
the opioid receptor activity, 37 and might be responsible
for the reduction of the efficacy of morphine's antino-
ciceptive effect in either opioid tolerance or neuropathic
pain. PTX ADP-ribosylation disrupts the Gi/Go protein-
coupled signal transduction, may lead to a predomin-
ance of the spinal excitatory receptor system.38,39 Many
studies have suggested that i.t. PTX-induced hyperal-
gesia results from a direct central mechanism, which
differs from the effect of peripheral nerve damage. 12,14
Animal and human studies had demonstrated a decreas-
ing analgesic effect of -opioid agonists on neuropathic
pain, and one of the mechanisms might be down-regula-
tion of functional -opioid receptors but not reduction of
receptor number.1,40 In chronic constriction injury (CCI)
of sciatic nerve rats, hyperalgesia was accompanied by a
rightward shift of opioid's antinociceptive dose response
curve.41 Similarly, we found that i.t. PTX injection pro-
duced a thermal hyperalgesia as well as a reduction of
morphine analgesic effect.42 Collectively, i.t. PTX injec-
tion can be used as an animal model to study the central
mechanisms of neuropathic pain syndrome. In combina-
tion with a microdialysis technique, a long-term CSF bio-
chemical changes can also be observed.
The Cellular Mechanisms of PTX-induced EAAs
Release
At least, three possible mechanisms are proposed
for the PTX-induced EAA release. Firstly, PTX inacti-
vates a G-protein, which is normally negatively regulat-
ing Ca2+ channels. Dolphin and Scott had demonstrated
that PTX enhanced the Ca2+ channel activity in dorsal
root ganglia.33 Secondly, PTX ADP-ribosylates Gi/Go
 ZHI-HONG WEN et al.
proteins, and thus increases the number of function of
Ca2+. Intracerebroventricular (i.c.v.) injection of PTX
increases the number of dihydropyridine (DHP) binding
sites (L-type calcium channel).43 Huston et al. also found
that PTX induced glutamate release from cerebellar
granule neurons via increasing in the presynaptic L-type
Ca2+-binding sites.35 Thirdly, PTX-induced EAAs re-
lease might be via PKC activation. Activation of Ca2+
channels results in an increasing of intracellular Ca2+ ,
which, in turn, results in Ca2+ -dependent glutamate ex-
ocytosis.44 All these changes may activate PKC and the
activated PKC increases NMDA receptors activity.45-47
In a study of NMDA-induced seizures, Durcan and Mor-
gan suggested that PTX induced glutamate release and
PKC activation were via increasing of intracellular Ca2+
concentration.36 PKC acts as a positive feedback to
increase EAAs release that is responsible for the neuronal
hypersensitivity and reduction of the nociceptive thres-
hold. The ability of PTX to increase EAA release in-
dicates that there is a tonic G-protein-mediated, accom-
panied with Ca2+ entry, inhibition of exocytotic process
in the presynaptic terminal.
PTX-induced EAAs Receptor Activation
Contributes to The Nociceptive Hypersensitization
Previous studies proposed that the development of
neuropathic pain syndrome is involved in EAAs release,
and subsequently would activate EAA receptors in dorsal
horn of the spinal cord. 7,48 In our study, we found that
this increase of aspartate and glutamate concentration
in the CSF after i.t. PTX injection was accompanied by
a thermal hyperalgesia, and it could be blocked by i.t.
NMDA antagonist D-AP5.42 Somers and Clemente also
demonstrated that chronic sciatic nerve constriction
injury induced neuropathic pain syndrome was accom-
panied by an increasing of EAAs content in the synapto-
some prepared from spinal cord dorsal horn. 49 Further-
more, Cui et al. found that an increase of basal release of
EAAs as well as a decrease of GABA release in spinal
dorsal horn was observed in rats following partial ligation
of the sciatic nerve.50 This elevation of EAAs likely
plays an important role in the development of painful
symptoms. The symptoms of allodynia and hyper-
algesia induced by i.t. injection of inflammatory agents
with accompaniment of EAAs release, were similar to
those observed in neuropathic pain syndrome.51 Ample
evidence has demonstrated that EAA receptors partici-
pate in the development and maintenance of neuropathic
pain syndrome, hyperalgesia and allodynia. Spinal ad-
ministration of NMDA or non-NMDA receptor antagon-
ists inhibits the neuropathic pain induced by sciatic nerve
ligation,52 spinal cord injury,53 or brachial plexus tran-
section.54 Collectively, from our previous reports, we
189
suggest that the PTX-induced EAAs release promotes
nociceptive signal transduction, presented as the thermal
hyperalgesia, by activating the pain facilitatory system,
thereby enhancing the nociceptive response and reducing
opioids' analgesic effect.42,55
The Effect of Inhibitory Amino Acids on
PTX-induced Neuropathic Pain Syndrome
Inhibitory neurotransmitter receptors (opioid,
GABA B, and A 1-adenosine) are linked with PTX-sensi-
tive inhibitory G-proteins, and inactivation of these in-
hibitory G-proteins may contribute to the activation of
excitatory receptors, such as the NMDA receptors. In
our recent study, PTX treatment was accompanied by a
decrease of CSF IAA glycine concentration, which might
unmask the excitatory receptors activity, and further
promote the development of thermal hyperalgesia.42
Glycine is a principal inhibitory neurotransmitter in the
spinal cord widely distributed in spinal dorsal horn, and
plays an inhibitory role in nociceptive transmission.56,57
Two major subtypes of glycine receptors (GlyRN and
GlyRA) had been demonstrated in rats. The neonatal-
type receptors (GlyRN) are only expressed in the spinal
cord of the newborn and replaced by the adult-type recep-
tors (GlyRA ) 2 weeks after birth, which exist only in the
spinal cord and the brain stem of adult animals.58 The in-
hibitory effect of glycine is different from its function as
the co-agonist with glutamate on the NMDA receptors.
Activation of glycine receptors results in chloride cur-
rents and produces a post-synaptic hyperpolarization.59
Numerous studies support that the function of glycine
in the spinal cord is related to nociceptive transmission.
Simpson et al. found that i.t. administration of glycine
and related compounds attenuated the neuropathic pain
syndromes of thermal and mechanical hyperalgesia. 60 A
reduction of glycine receptors was observed in the dorsal
horn of the spinal cord in chronic sciatic nerve constric-
tion-injured rats.61 Furthermore, Ishikawa et al. demon-
strated that i.t. injection of the glycine receptor antagonist
strychnine caused touch-evoked allodynia. 62 Moreover,
they found that this allodynia was reversed by NMDA
antagonists, and suggested that loss of glycine inhibition
might facilitate the release of EAAs, and then enhance
the post-synaptic excitability of the EAAs system.62 In
our study, we demonstrated that i.t. PTX injection, at
doses of 1 and 2 g, resulted in a decreasing of spinal gly-
cine level between days 2 and 8, and this reduction corre-
lated with the development of thermal hyperalgesia.42
Moreover, in the same study, we also found that glycine
administration inhibited this PTX-induced thermal hy-
peralgesia.42 A significant increase in the aspartate/
glycine and aspartate/GABA ratios is reported in clinical
patients with neuropathic pain state.63 Woolf and Man-
190 NEUORPATHIC PAIN AND PERUTSSIS TOXIN
nion had demonstrated that peripheral nerve injury re-
duced the number of inhibitory GABA neurons and
GABA and glycine receptors in the dorsal horn of the
spinal cord.3 Taken together, disinhibition of the inhibi-
tory receptor system may upset the balance between ex-
citatory and inhibitory receptors in the spinal cord, thus
resulting in EAA release, and hyperalgesia.
The Possible Role of NO in PTX-induced Thermal
Hyperalgesia
Glutamate is known to activate NMDA receptors
and results in activation of the NMDA-NO cascade
which is believed to contribute to the induction of neu-
ronal wind-up and central sensitization in the spinal cord.
Kitto et al. found that systemic or i.t. administration of
the NOS inhibitor, L-NAME, inhibited NMDA-induced
hyperalgesia.64 Wiertelak et al. suggested that the spinal
NMDA-NO cascade is involved in central hyperalgesia
development. 65 Furthermore, an up-regulation of NOS
expression is seen after sciatic nerve transection.66 We
also demonstrated that NOS inhibitor prevents the de-
velopment of neuropathic pain syndromes.54 I.t. injec-
tion of L-arginine was shown to produce thermal hyper-
algesia.67 Pretreatment with NO scavenger hemoglobin
delayed the development of thermal hyperalgesia in rats
with sciatic nerve constriction injury.68 These findings
suggest that NO also plays a role in thermal hyperalgesia
following nerve injury. However, in our recent study,
we failed to observe any changes in either NO release or
expression of NOS isoforms in the spinal cord in PTX-
rats.42 Moreover, Calcutt and Chaplan also failed to
demonstrate the effect of L-NAME on allodynia in dia-
betic or nerve-injured rats. 69 Luo et al. found that tight
ligation of spinal L5/L6 roots in Harlan Sprague-Dawley
and Holtzman rats increased nNOS mRNA levels in the
dorsal root ganglion, but not in the dorsal horn of the spi-
nal cord. 70 In the same study, they found that mechanical
allodynia was observed in Harlan Sprague-Dawley rats
after nerve ligation, and systemic nNOS inhibitor 7-nitro-
indazole (7-NI) treatment failed to prevent or reverse
the allodynia. Collectively, we suggest that NOS in the
dorsal horn of the spinal cord may indirectly contribute to
the neuropathic pain development.
Effect of PKC Isoforms on PTX-induced Thermal
Hyperalgesia
In our recent study, we found that PTX treatment
increased spinal PKC expression, but not , I, II iso-
forms.71 In contrast to the PKC , I, and II isoforms
which are widely distributed in the superficial dorsal
horn, the PKC isoform is expressed mainly in the inner
part of laminae II of spinal cord.72 Palecek et al. had de-
monstrated that PKC is expressed only in the excitatory
intemeurons, but not inhibitory interneurons in rat spinal
cord, and they suggested that PKC is a potential candi-
date contributing to the spinal nociceptor hypersensitiza-
tion.73 In PKC knock-out mice, a significant reduction
in tactile allodynia was observed after partial sciatic
nerve ligation.74 Moreover, an up-regulation of PKC
protein expression was also observed in neuropathic pain
animals after loose ligation of sciatic nerve.75 In our re-
cent study, we provided an additional evidence that i.t.
PTX-induced PKC up-regulation and translocation, via
NMDA receptor activation, might be responsible for the
development of thermal hyperalgesia,71 and thus we sug-
gested that the increased PKC content in the dorsal horn
of the PTX-treated rat spinal cord might be a conse-
quence of NMDA receptor activation, which induced
up-regulation and translocation of PKC from the cytosol
to the plasma membrane. This PKC up-regulation/
translocation modifies synaptic transmission in the spinal
dorsal horn, and contributes to the nociceptive hypersen-
sitization.
The Role of NMDA-PKC Signaling in Reduction of
Opioid's Analgesic Effect
Many studies had demonstrated that activation of
NMDA-PKC signal transduction cascade played an im-
portant role in neuropathic pain syndrome.52,76,77 Chen
and Huang had demonstrated that activation of PKC
down-regulated opioid receptor activity. 45 PKC activa-
tion was found to phosphorylate the -receptor-coupled
G-protein, thereby decreasing opioids' ability to inhibit
adenyl cyclase with resultant loss of morphine's antino-
ciceptive effect. 78 Activation of NMDA receptors and
subsequent PKC activation reduces the effectiveness of
morphine's antinociception in PTX-rat, via uncoupling
of G-protein from opioid receptors and disrupting the
extracellular signaling into the cells. 71,79 In Shen and
Grain's theory, activation of opioid receptors may ac-
tivate either the inhibitory Gi/Go or the stimulatory Gs-
proteins.80,81 Turning on the Gs-coupled opioid receptor
signal transduction may increase the PKC activity and
attenuate morphine's antinociception effect. I.t. PTX
breaks the balance between opioid receptor coupled in-
hibitory and excitatory G-proteins, in which the stimula-
tory Gs-protein signal transduction pathway becomes
prominent and results in an increasing of EAAs release,
and thus reduction of morphine's analgesic effect. The
up-regulation of excitatory opioid effect by PTX treat-
ment might play an important role in the reduction of
opioid's analgesic effect. Similarly, in our previous study,
we found that the PTX-induced thermal hyperalgesia was
accompanied by an increase of PKC expression in both
the synaptosomal membrane and cytosolic fractions of
 ZHI-HONG WEN et al.
the rat lumbar spinal dorsal horn and both effects were
inhibited by NMDA receptor antagonist D-AP5.71 In the
same study, the thermal hyperalgesia was accompanied
by an increasing of glutamate and aspartate levels in spi-
nal CSF dialysates, and it was attenuated by PKC inhibi-
tor chelerythrine. Chelerythrine not only prevented the
PTX-induced thermal hyperalgesia and CCI-induced
neuropathic pain but also acutely reversed morphine's an-
algesia in tolerant rats. 76,82 Recently, we found that PKC
inhibitor chelerythrine was able to reverse the reduction
of morphine's antinociceptive effect, while chelerythrine
itself did not produce any antinociceptive effect in naive
animals.42 Similar to our study. Smith et al. found that
non-selective PKC inhibitors Go-7874 and sangivamycin
had no effect on morphine's antinociception in naive
mice, but could reverse morphine's antinociception in
morphine tolerant rats.82 The evidence suggests that
PKC may directly down-regulate opioid receptor func-
tion, and thus results in a reduction of opioid's analgesic
effect. The rationale for the involvement of NMDA-PKC
signaling for the poor response of opioids to neuropathic
pain is that the increasing of pre-synaptic EAAs release
and activation of post-synaptic NMDA receptors, initi-
ated by PTX treatment, may trigger PKC activation, and
thus reducing opioid's analgesic effect. This positive
feedback regulation of NMDA-PKC cascade is involved
in the enhancement of opioid receptor phosphorylation
and the reduction of opioid receptor's function. 45 Taken
together, our data suggest that PTX-induced thermal hy-
peralgesia and the lose of morphine's analgesia share a
common cellular mechanism, at least in part, activation
of the EAAs-PKC system. The Ca2+ -regulated intracel-
lular PKC is likely a linkage between hyperalgesia and
reduction of opioid's analgesic effect in PTX-rats.
Summary
From our serial studies, we found that i.t. adminis-
tration of PTX induced thermal hyperalgesia in rats,
which was accompanied by a prolonged increase in
EAAs concentration and a decrease in IAA glycine con-
centration in spinal CSF dialysates. The changes in CSF
aspartate, glutamate, and glycine concentrations were
paralleled to the thermal hyperalgesia development. Fur-
thermore, we found an up-regulation/translocation of spi-
nal PKC after i.t. PTX treatment. Infusion of NMDA
antagonist D-AP5 prevented both thermal hyperalgesia
and increase of PKC isoform expression in PTX-treated
rats. I.t. PTX treatment did not affect NO concentration
in the CSF as well as the nNOS or iNOS protein ex-
pression. The PTX-induced hyperalgesia was reversed
by i.t. NMDA antagonist D-AP5, glycine and PKC in-
hibitor chelerythrine. Pretreatment with PKC inhibitor
191
chelerythrine l h prior to morphine challenge on day 5
after PTX treatment could prevent the morphine-preci-
pitated spinal glutamate and aspartate release and reverse
morphine's analgesic effect in PTX-rats. These results
suggest that PTX-induced neuropathic pain syndromes
are involved in EAAs, IAAs and PKC alteration. The
cellular mechanisms of PTX-induced spinal nociceptive
hypersensitization are depicted diagrammatically in Fig.
1. (1) PTX is able to ADP-ribosylate the post-synaptic
opioid receptor coupled -subunit of inhibitory G-pro-
teins (Go/Gi). It disrupts the inhibitory Gi/Go protein-
coupled signal transduction (such as -receptors), thus
breaks the balance between Gs and Gi protein signal
transduction, leading to a predominance of the spinal ex-
citatory receptor system. (2) PTX inhibits the activation
of -opioid receptor signal transduction (opening Gi pro-
tein-gated potassium channels and closing voltage-gated
calcium channels). (3) PTX also presynaptically induces
the release of glutamate (Glu) and aspartate (Asp), via
exocytosis by modulating the L-type Ca2+ channel activ-
ity at the pre-synaptic nerve terminals. (4) The released
glutamate and aspartate activate postsynaptic NMDA re-
ceptors and open the NMDA ligand-gated Ca2+ channel
with increases of Ca2+ influx into postsynaptic neurons,
and (5) followed by activation of PKC- isoform. (6) A
transient increase in NO production via activating the
NOS or PKC activity. (7) Inactivation of PKC by PKC
inhibitor chelerythrine inhibits PTX-induced nociceptive
hypersensitization. As we know, extracellular EAAs re-
leased from the pre-synaptic nerve terminals are counter-
balanced by glutamate transporters in neurons/glial cells
which terminate the glutamatergic signaling, and thus
protecting neurons from EAAs toxicity. (8) Activation
of PKC was found to down-regulate the glutamate trans-
porter activity and expression, which results in an ac-
cumulation of synaptic EAAs concentration. (9) PKC
phosphorylates the -receptor-coupled G-protein and
results in down-regulate opioid receptor activity. (10)
Activated PKC results in an enhanced release of EAAs,
and it further excites the spinal nociceptive responses.
(11) Decreasing of glycine release results in a reduction
of post-synaptic strychnine sensitive chloride channel
(GlyR) flow and subsequently decreasing of the inhibi-
tory mechanisms, thereby, disrupting the balance be-
tween inhibitory and excitatory receptor system. (12)
NO, a reactive, diffusible, and short-lived neuromodula-
tor may act retrograde as an initiator to provide a positive
feedback regulation of pre-synaptic neuron activity.
From this review, we know that activation of EAA
receptors, particularly the NMDA receptors, are involved
in the PTX-induced neuropathic pain. Glutamate level
released from nerve terminals in the synaptic junction
is counterbalanced by glutamate transporters in neurons
and glial cells, which terminate the glutamatergic signal-
192 NEUORPATHIC PAIN AND PERUTSSIS TOXIN
Fig. 1. The schematic diagram of the hypothetic cellular mechanisms of PTX-induced neuropathic pain syndrome.
ing, and thus protecting neurons from the glutamate tox-
icity and neuroplasticity. In our recent study, we found
that the PTX-induced thermal hyperalgesia may be re-
lated to the PKC,71 whose activation was found to down-
regulate the glutamate transporter activity and ex-
pression,83,84 and phosphorylation of NR2B subunit of
the NMDA receptors.85 Therefore, examining the role
of glutamate transporters and NMDA receptors pho-
sphorylation in the behavior expression of PTX-rats
may be another research direction for the neuropathic
pain management.
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196 NEUORPATHIC PAIN AND PERUTSSIS TOXIN

(neuropathic pain)
(hyperalgesia) (allodynia)

(thermal hyperalgesia)
Gi Go ADP-ribosylation





(glutamate) (aspartate) (glycine)
(serine) (asparagine) (taurine)
(arginine)
NMDA D-AP5
C (protein kinase C) chelerythrine
C-
I II (isoenzyme) C-
D-AP5 D-AP5 C- I
II

C
C
( ) C

Protein kinase C