The Journal of Neuroscience, September 3, 2003, 23(22):8098-8108
Previous Article | Next Article 
Role of Thalamic Phospholipase C
4 Mediated by Metabotropic Glutamate Receptor Type 1 in Inflammatory Pain
Mariko Miyata,1,2
Hideki Kashiwadani,2
Masahiro Fukaya,3
Takayuki Hayashi,2
Dianqing Wu,4
Tutomu Suzuki,5
Masahiko Watanabe,3 and
Yoriko Kawakami2
1Department of Information Physiology, National
Institute for Physiological Sciences, Okazaki 444-8585, Japan,
2Department of Physiology, Tokyo Women's Medical
University, Tokyo 162-8666, Japan, 3Department of
Anatomy, Hokkaido University, School of Medicine, Hokkaido 060-8638, Japan,
4Department of Genetics and Developmental Biology,
University of Connecticut, Farmington, Connecticut 06030, and
5Department of Toxicology, Faculty of Pharmaceutical
Science, Hoshi University, Tokyo 142-8501, Japan
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Abstract
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Phospholipase C (PLC) 
4, one of the four isoforms of PLCs, is
the sole isoform expressed in the mouse ventral posterolateral thalamic
nucleus (VPL), a key station in pain processing. The mouse thalamus also has
been shown to express a high level of metabotropic glutamate receptor type 1
(mGluR1), which stimulates PLCs through activation of G
q/11
protein. It is therefore expected that the thalamic mGluR1-PLC4 cascade
may play a functional role in nociceptive transmission. To test this
hypothesis, we first studied behavioral responses to various nociceptive
stimuli in PLC4 knock-out mice. We performed the formalin test and found
no difference in the pain behavior in the first phase of the formalin test,
which is attributed to acute nociception, between PLC4 knock-out and
wild-type mice. Consistent with this result, acute pain responses in the hot
plate and tail flick tests were also unaffected in the PLC4 knock-out
mice. However, the nociceptive behavior in the second phase of the formalin
test, resulting from the tissue inflammation, was attenuated in PLC4
knock-out mice. In the dorsal horn of the spinal cord where PLC1 and
PLC4 mRNAs are expressed, no difference was found between the wild-type
and knock-out mice in the number of Fos-like immunoreactive neurons, which
represent neuronal activity in the second phase in the formalin test. Thus, it
is unlikely that spinal PLC4 is involved in the formalin-induced
inflammatory pain. Next, we found that pretreatment with PLC inhibitors,
mGluR1 antagonists, or both, by either intracerebroventricular or
intrathalamic injection, attenuated the formalin-induced pain behavior in the
second phase in wild-type mice. Furthermore, activation of mGluR1 at the VPL
enhanced pain behavior in the second phase in the wild-type mice. In contrast,
PLC4 knock-out mice did not show such enhancement, indicating that
mGluR1 is connected to PLC4 in the VPL. Finally, in parallel with the
behavioral results, we showed in an electrophysiological study that the time
course of firing discharges in VPL corresponds well to that of pain behavior
in the formalin test in both wild-type and PLC4 knock-out mice. These
findings indicate that the thalamic mGluR1-PLC4 cascade is indispensable
for the formalin-induced inflammatory pain by regulating the response of VPL
neurons.
Key words: thalamus; phospholipase C4; metabotropic glutamate receptor type 1; formalin test; inflammatory pain; knock-out mouse; electrophysiology
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Introduction
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Our previous study demonstrated the characteristic expression patterns for
four isoforms of phosphoinositide-specific phospholipase C (PLC) s in
the mouse brain, with PLC4 mRNA, one of the major neuronal isoforms,
being predominantly expressed in the thalamus, including the ventral
posterolateral thalamic nucleus (VPL;
Watanabe et al., 1998
).
PLCs are activated by interacting with the subunit of the Gq
subclass of GTP-binding proteins
(Strathmann and Simon, 1990;
Wilkie et al., 1991;
Rebecchi and Pentyala, 2000).
The Gq-PLC cascade is engaged by the activation of hormone or
neurotransmitter receptors such as the bradykinin receptor and metabotropic
glutamate receptor types 1 and 5 (mGluR1 and mGluR5; the so-called group I
mGluRs; Masu et al., 1991;
Abe et al., 1992;
Haley et al., 2000).
PLCs produce a pair of second messengers, diacylglycerol and inositol
1,4,5-trisphosphate, which activate protein kinase C (PKC) and release
Ca2+ from intracellular stores, respectively
(Exton, 1996). This cascade
via mGluR1 underlies various brain functions such as the elimination of
climbing fiber 
Purkinje cell synapses, cerebellar long-term depression
(Kano et al., 1998;
Miyata et al., 2001), and
motor learning (Ichise et al.,
2000; Kishimoto et al.,
2001).
A number of molecular mechanisms that contribute to pain processing have
recently been clarified at the peripheral tissue
(Bhave et al., 2001;
Zhou et al., 2001) and spinal
levels (Willis and Coggeshall,
1991). Group I mGluRs have been found to modulate inflammatory
pain at the dorsal horn of the spinal cord via activation of their second
messenger and protein kinases (Coderre,
1992; Fisher and Coderre,
1996; Young et al.,
1997; Karim et al.,
2001). Most studies on modulatory molecules in the afferent pain
pathway have focused on their involvement at the peripheral and spinal levels,
whereas very few studies have implicated the role of glutamate
receptor-interacting molecules in pain sensation at the supraspinal level. The
NMDA receptor contributes to nociceptive responses and hyperalgesia associated
with neurogenic inflammation (Eaton and
Salt, 1990; Kolhekar et al.,
1997) in the VPL of the thalamus, which receives spinothalamic
input (Lund and Webster, 1967;
Craig and Burton, 1981;
Cliffer et al., 1991),
responds to various noxious stimuli
(Peschanski et al., 1983;
Yokota et al., 1988;
Willis and Westlund, 1997),
and relays nociceptive information to the cerebral cortex
(Willis, 1985). It is reported
that metabotropic glutamate receptors play roles in nociceptive responses in
the rat VPL (Salt and Eaton,
1994). Forebrain NMDA receptor 2B, one of the subunits of NMDA
receptors, is involved in inflammatory pain behavior but not in acute pain
behavior in transgenic mice (Wei et al.,
2001). The second messenger and downstream kinases from these
receptors are presumably linked to pain behavior at supraspinal levels.
Because the mouse VPL expresses a high level of mGluR1
(Shigemoto and Mizuno, 2000),
the thalamic mGluR1-PLC4 cascade may be involved in modulating pain
processing. To clarify this possibility, we studied behavioral and
electrophysiological responses to the formalin test in mice lacking the
PLC4 gene. We also studied the effects of PLC
inhibitors, mGluR1 antagonists, and a group I mGluR agonist on the
formalin-induced pain behavior at the supraspinal and thalamic levels. Our
results indicate that the mGluR1-PLC4 cascade in the mouse thalamus is
essential for inflammatory pain processing induced by formalin injection.
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Materials and Methods
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Animals. Mice (PLC4-/- and PLC4+/+) of either sex
(7-15 weeks old) were used in the study. They were generated with a CJ7 ES
cell clone derived from 129sv mice. The generation of PLC4 knock-out
mice has been described in detail elsewhere
(Jiang et al., 1996;
Kano et al., 1998). The
founder mice were then backcrossed with C57BL/6J mice. Knockout mice of F4 and
later generations were compared with corresponding wild-type littermates used
as controls.
Behavioral assays. All animal experiments were conducted in
accordance with the guidelines of the National Institute of Mental Health
Animal Care and Use Committee.
Formalin test. Thirty microliters of formalin (5%) were
administered subcutaneously into the plantar of the left hindpaw. The
cumulative duration of licking and lifting of the injected paw was measured
every 5 min immediately after the injection. Two hours after the injection,
the mouse was deeply anesthetized and perfused with saline followed by 4%
paraformaldehyde. The spinal cord was removed and postfixed for c-Fos
immunohistochemistry (see below). All statistical analyses were performed
using the repetitive ANOVA, with comparison between the experimental groups
and the control groups at each time using Dunnett's post hoc
test.
Thermal pain assay. The hot plate test was performed using an
electronically controlled hot plate (MK-350A; Muromachi Instrument, Tokyo,
Japan) heated between 42 and 58°C. The latency before the animal licked
its hindpaw or jumped on the hot plate was recorded. The cutoff time was 50
sec for 52°C and 30 sec for temperatures of >55°C. The tail flick
test was performed using a commercially available apparatus (MK-330A;
Muromachi Instrument) consisting of an irradiator for heat stimulation and a
photosensor for detection of the tail flick behavior. The latency from the
start of irradiation to the tail flick reaction was measured. To prevent the
mouse tails from being injured, the cutoff time was determined to be 10 sec.
The tail flick reaction was measured two times in one test, and the average
was considered as the latency. All statistical analyses were performed using
one-way ANOVA.
Tissue preparation and immunohistochemistry. To visualized Fos
expression in the dorsal horn of the spinal cord, the animals (wild-type and
PLC4 knock-out mice) were perfused with 100 ml of normal saline,
followed by 100 ml of ice-cold 4% paraformaldehyde in 0.1 M
phosphate buffer (PB), pH 7.4. Then, the spinal cord was removed and postfixed
at 4°Cin0.1 M PB with 30% sucrose overnight for cryoprotection.
Coronal sections were cut at 20 µm thickness and collected on silane-coated
glass slides. For Fos protein staining, a polyclonal anti-rabbit c-Fos
antibody (Ab-5, 1:20,000; Oncogene Product) was used. The ABC method and then
DAB staining for visualization were performed as described previously
(Imaki et al., 2001). To
quantify Fos-like immunoreactive (Fos-LI) neurons, we counted neurons with
stained nuclei from laminae I and II, III and IV, and V and VI in the lumbar
4-5 dorsal horn. Statistical analysis of the data were performed by one-way
ANOVA for the different groups of animals.
To visualize expression patterns of PLC isoforms in the mouse spinal
cord, thalamus, and forebrain, fresh-frozen coronal sections of C57BL/6J adult
mice were used. Antisense oligonucleotides for mouse PLC1-4 mRNAs were
synthesized, and in situ hybridization histochemistry was performed
as reported previously (Watanabe et al.,
1998).
Drug injections. Intracerebroventricular injection of 4 µl of a
drug was administered under light halothane anesthesia as described by
Sanchez-Blazquez et al. (1995)
using a 10 µl microsyringe with a 31 gauge needle. Injections were
unilateral and performed on the same side. At the end of each experiment,
black ink was injected through the same route to ensure that the initial
intracerebroventricular injection was successful. For intrathalamic injection,
a mouse was initially deeply anesthetized with sodium pentobarbital (Nembutal,
50 mg/kg of body weight) and then placed on a stereotaxic frame. A stainless
steel guide cannula (26 gauge blunted needle) was then inserted vertically
above the right VPL. The guide cannula was positioned 1.2 mm posterior to the
bregma, 1.85 mm lateral to the midline, and 2.7 mm in depth from the surface
of the brain and was secured with dental resin. Seven days after the guide
cannula was placed, microinjections were performed in awake animals through a
stainless steel injection cannula (outer diameter, 0.2 mm; inner diameter,
0.08 mm) inserted into the guide cannula. The cannula was connected to a 1.0
µl microsyringe via polyethylene tubing filled with distilled water. The
drugs or vehicles were administered in a volume of 0.2 µl. Before starting
a series of experiments, we confirmed the extent of diffusion in the thalamus
by injecting 0.2 µl of 10% fast blue solution through the cannula in mice.
Diffusion of 
600-700 µm in diameter, including the VPL, was observed,
indicating that the drug remained in the ipsilateral thalamus.
The drugs used in the present study were 1-(6-[(17-methoxyestra-1,3,5
[10]-trien-17-yl) amino] hexyl]-1H-pyrrole-2,5-dione (U73122
[GenBank]
; Calbiochem, La
Jolla, CA),
1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphorylcholine
(Et-18-OCH3; Calbiochem),
1-(6-[-([17]-3-methoxyestra-1,3,5[10]-trien-17yl)amino]hexyl)-25-pyrrolidinedione
(U-73343; Sigma-Aldrich), (RS)-1 aminoindan-1,5-dicarboxylic acid
(AIDA; Tocris, Bristol, UK), 7-(hydroxyimino) cyclopropa [b]
chromen-1acarboxylate ethyl ester (CPCCOEt; Tocris),
(RS)-3,5-dihydroxyphenylglycine [(RS)-DHPG; Tocris], and
2-methyl-6-(phenylethynyl) pyridine hydrochloride (MPEP hydrochloride;
Tocris). They were dissolved in artificial CSF (ACSF), except for U73122
[GenBank]
,
which was dissolved in ACSF with 0.4% DMSO. ACSF contained (in mM):
125 NaCl, 2.5 KCl, 2-2.5 CaCl2,1-1.3 MgSO4, 1.25
NaH2PO4, 26 NaHCO3, and 20 glucose.
Intracerebroventricular or thalamic injection of ACSF with 0.4% DMSO caused no
significant difference in the spontaneous and nociceptive behaviors compared
with that with the injection of ACSF alone.
Electrophysiology. Wild-type and PLC4 knock-out mice
weighing 25-35 gm were anesthetized with urethane (1.2 gm/kg, i.p.). The
rectal temperature was maintained between 37 and 38°C with a heating pad.
The depth of anesthesia was monitored by assessing the animals' responsiveness
in terms of eye blinks. A mouse was mounted on the stereotaxic holder without
prevention of free respiration. The head position was adjusted until the
heights of the lambda and bregma skull points were equal. A small window was
opened at the dorsal surface of the skull, and a stainless steel electrode
(9-12 M
) was inserted into the VPL of the thalamus. The coordinates
used for recordings in the VPL were 1.2-1.3 mm caudal and 1.8-1.9 mm lateral
to the bregma and 2.6-3.5 mm below the cortical surface. Receptive fields of
thalamic neurons were first identified by their spike discharges in response
to a gentle pinch with forceps and a heating probe (25-58°C; DPS-777J; Dia
Medical System, Tokyo, Japan). Activities of thalamic neurons with receptive
fields in the contralateral hindpaw were then examined in the formalin test. A
30 gauge needle that was attached to a syringe pump via a silicone tube (10 cm
long) was inserted subcutaneously and maintained in place for at least 5 min
to eliminate the effect of needle insertion. A bolus (30 µl) of 5% formalin
was then injected within 3 sec, and the needle was then immediately removed.
Responses of VPL neurons to formalin injection were continuously recorded from
1 min before to 1 hr after the injection. Spike discharges were amplified with
a bandpass filter (AVH-11; Nihon Kohden, Tokyo, Japan) at 0.5-10 kHz. The
recorded signals were sampled at 10 kHz with an analog-to-digital converter
(CED 1401 Plus; Cambridge Electronic Design, Cambridge, UK), and stored in a
Pentium personal computer for further off-line analyses (see below).
After each recording session, the recording site was marked by passing a
current (10 µA, 20 sec duration) though the electrode. After all recording
sessions were completed, the mouse was perfused intracardiacally with 4%
paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. Brain sections
of 50 µm thickness were prepared. After reaction with 1% potassium
ferrocyanide and 1% HCl solution, each marked point was visualized as a small
blue spot. The VPL was identified by cytochrome oxidase staining of the same
sections. We thereby confirmed that recording sites were located in the
VPL.
Off-line analyses. Single-unit discharges were isolated from
recorded signals by template-matching procedures using Spike 2 (Cambridge
Electronic Design). Firing rate histograms were plotted with a bin width of 1
sec. The average firing rate with its SD 1 min before formalin injection was
considered the basal activity. A neuron was regarded as responding to the
stimulus when its firing rate after formalin injection exceeded the basal
activity. To evaluate the duration of the response in the early phase (0-5 min
after the injection) and that in the late phase (15-60 min after the
injection), the numbers of bins during the two phases in which the firing rate
exceeded the basal activity were counted. All statistical analyses were
performed using the unpaired Student's t test.
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Results
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Behavioral studies in wild-type and PLC4 knock-out mice
PLC4 knock-out and wild-type mice were subjected to several
nociception tests. We first performed the formalin test in the wild-type and
knock-out mice. Injection of 5% formalin subcutaneously into the hindpaw of
wild-type mice resulted in a typical biphasic nociceptive response
(Tjolsen et al., 1992). The
first phase, usually lasting within 5 min, occurred immediately after formalin
injection and was characterized by intense licking and lifting of the injected
paw. The second phase, also characterized by licking and lifting of the
injected paw, occurred 15-20 min after formalin injection and lasted for
60 min. The first phase of the formalin test is commonly attributed to
acute nociception occurring in direct activation of nociceptive fibers
(Puig and Sorkin, 1996),
whereas the second phase is attributed to tonic nociception resulting from
tissue inflammation. There was no difference
(Fig. 1) in the duration of the
first phase of the pain response to the injection measured within the first 5
min between the knock-out and wild-type mice: 148.8 ± 16.7 sec (mean
± SEM; n = 10) and 144.8 ± 18.1 sec (n = 10),
respectively. In contrast, the mean duration of the pain behavior in the
second phase (15-45 min after formalin injection) was significantly attenuated
to 41.5% (p < 0.01; Fig.
1) in the knock-out mice (334.8 ± 62.1 sec) compared with
that in wild-type mice (803.6 ± 46.2 sec). The knock-out mice showed
normal edema. There was no significant difference in the mean thickness at the
site of the formalin-injected paw between the wild-type mice (3.6 ±
0.11 mm, mean ± SD; n = 9) and knock-out mice (3.8 ±
0.12 mm; n = 9) 2 hr after the injection, indicating that the
inflammatory change at the injected site in the knock-out mice was the same as
that in the wild-type mice.
Because PLC4 knock-out mice showed no alternation of pain behavior in
the first phase, we performed other acute pain assays. The tail flick test was
used to measure spinal pain reflexes, and the hot plate test was applied to
measure pain responses involved at the supraspinal level
(Chapman et al., 1985). We
found no difference in withdrawal responses between PLC4 knock-out and
wild-type mice in the tail flick test (Fig.
2A). We also observed no difference in the withdrawal
latencies in the hot plate test at various temperatures
(Fig. 2B).
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Figure 2. Acute pain responses to thermal stimulus in the tail flick test
(A) and hot plate test (B). No significant differences in
the latencies of withdrawal (mean ± SEM) from thermal stimuli were
observed between wild-type mice (open bars) and knock-out mice (filled bars)
in the tail flick test (n = 19) or the hot plate test (n =
19).
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These results indicate that lack of PLC4 reduces the formalin induced
inflammatory pain behavior, whereas it does not influence on the acute pain
behavior.
Expression of PLC isoforms in the somatosensory system
To identify the site among multiple somatosensory stations involved in the
attenuation of pain behavior in the second phase in the PLC4 knock-out
mice, we investigated the expression patterns of PLC isoforms in the
spinal cord, thalamus, and somatosensory cortex
(Fig. 3). In the spinal dorsal
horn of adult wild-type mice, both PLC1 and PLC4 mRNAs were
expressed (Fig. 3A-D).
In the VPL of the thalamus, PLC4 mRNA was the sole isoform transcribed
(Fig. 3E-H),
consistent with our previous report
(Watanabe et al., 1998). In
somatosensory cortex S1, PLC1 mRNA was expressed at high levels together
with PLC4 mRNA at a low level (Fig.
3E,H). In particular, PLC1 mRNA was the sole
isoform transcribed in layer IV of the somatosensory cortex, a recipient layer
for thalamocortical fibers. PLC2 and PLC3 mRNAs were almost
undetectable in the spinal cord, thalamus, and somatosensory cortex.
On the basis of these results, PLC4 is the sole isoform of PLCs
at the thalamic relay station of the VPL, whereas PLC1 and PLC4
are both expressed in the spinal cord. In the somatosensory cortex, PLC1
is the sole isoform in layer IV; both PLC1 and PLC4 are expressed
in the remaining layers.
Fos-LI neurons in the dorsal horn of the spinal cord in the formalin
test
Because no difference was found in edema size of the injected paw between
the wild-type and knock-out mice, it is reasonable to consider that similar
inflammatory inputs were conveyed to the dorsal horn of the spinal cord. The
observation that PLC4 co-localized with PLC1 is expressed in the
dorsal horn raises the possibility that lack of spinal PLC4 may
contribute to the attenuation of pain behavior in the second phase in the
knock-out mice. To examine this possibility, we counted the number of Fos-LI
neurons in all layers of the ipsilateral dorsal horn 2 hr after the
administration of formalin in PLC4 knock-out and wild-type mice
(Hunt et al., 1987). The
number of Fos-LI neurons corresponds to spinal neuronal activity during the
second phase (Abbadie et al.,
1992,
1997;
Todd et al., 1994;
Malmberg et al., 1997). The
highest number of Fos-LI neurons induced by formalin injection was found in
layers I and II of lumbar segment 4-5in both knock-out (102.5 ± 7.5)
and wild-type (98 ± 10.6) mice (Fig.
4A,B). Numbers of FOS-LI neurons had no significant
differences in all layers of the dorsal horn between the wild-type and
knock-out mice (Fig.
4C).
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Figure 4. Fos-LI neurons of the dorsal horn of the spinal cord after formalin
injection in knock-out and wild-type mice. A, B, Fos-LI cells in the
dorsal horn of the spinal cord ipsilateral to the formalin injection site in
wild-type mice (A) and PLC4 knock-out mice (B). Scale
bar, 50 µm. C, Numbers of Fos-LI neurons (mean ± SD) in
laminae I and II, III and IV, and V and VI of the ipsilateral spinal cord of
wild-type and knock-out mice injected with saline (Control: wild type,
n = 9; knock-out, n = 6) or with formalin (Formalin treated:
wild type, n = 9, knock-out, n = 6) into the hindpaw.
**p < 0.01 compared with the wild-type and knock-out controls,
respectively.
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Intracerebroventricular injection of PLC inhibitor in the formalin
test
To test that PLC4 modulates inflammatory pain processing at the
supraspinal level, U73122
[GenBank]
, a potent inhibitor of PLC
(Bleasdale et al., 1990), was
injected (0.5 or 5.4 nmol/mouse.) into the lateral cerebral ventricle of
wild-type mice, and the formalin test was performed 10 min after the
injection. Pain behavior in the first phase was not significantly different
between U73122
[GenBank]
- and vehicle-injected mice regardless of the dose of U73122
[GenBank]
(Fig. 5A,C), with mean
durations ± SEM of pain behavior in the first phase of 137.3 ±
28.7 sec for vehicle-injected mice (n = 8) and 124.9 ± 25.0
sec for the 5.4 nmol U73122
[GenBank]
-injected mice (n = 8). In contrast, pain
behavior in the second phase was attenuated in the U73122
[GenBank]
-injected mice in a
dose-dependent manner compared with the vehicle-injected mice
(Fig. 5A,C). When 5.4
nmol of U73122
[GenBank]
was injected, the mean duration of the pain behavior in the
second phase decreased to 19.4% (88.2 ± 24.2 sec; n = 8;
p < 0.01) compared with that in the vehicle-injected mice (458.4
± 40.2 sec; n = 8).
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Figure 5. Attenuation of the second phase of the formalin-induced nociceptive
behavior in a dose-dependent manner after intracerebroventricular injection of
U73122
[GenBank]
and AIDA. Time courses after 10 min of pretreatment with 5.4 nmol of
U73122
[GenBank]
(n = 8; A), 100 nmol of AIDA (n = 8;
B) and 100 nmol of MPEP (n = 5; B) compared with
those after vehicle injection (n = 8) are shown. Each data point
represents the mean duration ± SEM. **p < 0.01 compared
with the vehicle injection. C, D, Dose dependence curves for the
effects of U73122
[GenBank]
(C) and AIDA (D) injection on the first
phase (within the first 5 min after formalin injection; open squares) and the
second phase (cumulative duration of the pain behavior between 15 and 45 min;
filled circles), respectively. Each data point represents the mean duration
± SEM. **p < 0.01 compared with the vehicle
injection. The number of animals is indicated for each point.
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Intracerebroventricular injection of mGluR1 antagonist in the
formalin test
PLC4 is known to be activated by group I mGluRs
(Kim et al., 1997). mGluR1 is
highly expressed in the mouse thalamus, whereas mGluR5 is only weakly
expressed in this region. First, to examine whether mGluR1 is also involved in
the inflammatory pain processing at the supraspinal level, a formalin test was
performed 10 min after pretreatment with AIDA (10 or 100 nmol/mouse), an
antagonist of mGluR1, injected into the cerebral ventricle in wild-type mice.
The doses of AIDA adopted were based on a report by Moroni et al.
(1997). The
intracerebroventricular injection of AIDA attenuated formalin-induced pain
response in the second phase in a dose-dependent manner. When 100 nmol of AIDA
was injected, the mean duration of the pain behavior in the second phase
decreased to 18.8% (86.2 ± 11.7 sec; n = 8; p <
0.01; Fig. 5B,D),
compared with vehicle injection (458.4 ± 40.2 sec; n = 8).
AIDA had no effect on the pain behavior in the first phase, the mean durations
of pain behavior in the first phase being 137.2 ± 22.1 sec in the case
of vehicle injection (n = 8) and 158.9 ± 11.7 sec in the case
of 100 nmol AIDA injection (n = 8). Second, to test the contribution
of mGluR5, MPEP, a specific antagonist
(Gasparini et al., 1999), was
injected into the cerebral ventricle in the wild-type mice at 10 and 100 nmol
[the doses adopted were based on previous reports
(Chapman et al., 2000;
Berrino et al., 2001;
Fisher et al., 2002)]. No
significant change in the pain behavior was evident in the first phase
(increases in mean duration of only 5 and 8% with 10 and 100 nmol,
respectively; p > 0.05) or in the second phase (increases in mean
duration of only 3 and 2%; p > 0.05). The mean duration of pain
behavior was 147.8 ± 11.4 sec (n = 5) in the first phase and
467 ± 22.5 sec in the second phase in the case of 100 nmol MPEP
injection (Fig.
5B).
Thalamic injections of PLC inhibitors and mGluR1 antagonists
Because PLC4 is the sole isoform in the mouse VPL of the thalamus, we
studied whether thalamic PLC4 is involved in the formalin-induced pain
behavior by injecting 0.2 µl of 5.4 nmol U73122
[GenBank]
or 10 nmol Et-18-OCH3,
another selective inhibitor of PLC (Powis
et al., 1992), into the VPL of wild-type mice via a guided
cannula. These doses of the two drugs were based on a report by Narita et al.
(2000). The formalin test was
performed 10 min thereafter. The intrathalamic injection of U73122
[GenBank]
(5.4
nmol/mouse; n = 7) decreased the mean durations of the pain behavior
in the second phase to 30.5% (208.5 ± 32.1 sec) in comparison with the
intrathalamic vehicle injection (683.2 ± 53.5 sec; n = 12;
Fig. 6A). The
intrathalamic injection of U73343
[GenBank]
(5 nmol; n = 7), an inactive analog
of U73122
[GenBank]
(Bleasdale et al.,
1990), did not decrease the duration of pain behavior in the
second phase significantly (587.4 ± 72.4 sec; p > 0.5),
indicating that the effect of U73122
[GenBank]
was specific. Similarly, the
intrathalamic injection of Et-18-OCH3 (10 nmol/mouse; n = 7) 10 min
before formalin injection also decreased the mean durations of the pain
behavior in the second phase to 42.3% (289.0 ± 32.6 sec). Neither
thalamic injection of U73122
[GenBank]
nor that of Et-18-OCH3 had a significant effect
on the spontaneous behavior and pain behavior in the first phase (170.5
± 9.6 sec with U73122
[GenBank]
and 184.4 ± 8.6 sec with Et-18-OCH3) in
comparison with the vehicle (189 ± 8.5 sec) and U73343
[GenBank]
(192.6 ±
11.2 sec) injections.
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Figure 6. Attenuation of formalin-induced nociceptive behavior in the second phase
after thalamic injection with PLC inhibitors (A) and mGluR1
antagonists (B). A, Time course graphs after 10 min of
pretreatment with 5.4 nmol of U73122
[GenBank]
(n = 7) and 10 nmol of
Et-18-OCH3 (n = 7) compared with that for vehicle injection
(n = 12). B, Time courses after 10 min of pretreatment with
100 nmol of AIDA (n = 6) and 50 nmol of CPCCOEt (n = 9)
compared with that after vehicle injection (n = 12). Each data point
represents the mean time ± SEM. * p < 0.05;
**p < 0.01 compared with the vehicle injection.
|
|
Because PLC4 is well known to be activated by mGluR1
(Nakanishi, 1994), which is
also expressed in the mouse thalamus, we examined whether thalamic mGluR1 is
also involved in the formalin-induced pain behavior by injecting AIDA, an
antagonist of mGluR1, into the VPL. Similar to the results obtained from the
thalamic injection of PLC inhibitors, thalamic injection of 0.2 µl of AIDA
(100 nmol/mouse; n = 6), in comparison with the vehicle injection
(n = 8), had no effect on the pain behavior in the first phase (187.0
± 11.2 sec) but significantly decreased the mean durations of the pain
behavior in the second phase to 61.3% (418.9 ± 37.2 sec;
Fig. 6B). Furthermore,
we performed a similar experiment with thalamic injection of CPCCOEt, an
mGluR1-selective noncompetitive antagonist. The dose adopted was 50 nmol based
on the report by Maione et al.
(2000). Thalamic injection of
0.2 µl of CPCCOEt (n = 9) also had no effect on the mean duration
of the pain behavior in the first phase (174.2 ± 7.7 sec) but
significantly decreased the mean duration of the pain behavior in the second
phase to 27.9% (191.0 ± 32.0 sec;
Fig. 6B). Thalamic
injection of either 100 nmol of AIDA or 50 nmol of CPCCOEt alone did not
affect the spontaneous behavior of mice.
Thalamic injection of group I mGluR agonist in wild-type and
PLC4 knock-out mice
Given that pretreatment of the thalamus with either mGluR1 antagonists or
PLC inhibitors reduced the pain behavior in the second phase in wild-type
mice, it is surely expected that mGluR1 connecting to PLC4 in the
thalamus enhances the formalin-induced inflammatory pain. To confirm this,
intrathalamic injection of the group I mGluR agonist (RS)-DHPG (0.2
µl, 100 nmol/mouse) was performed 10 min before the formalin test in the
wild-type and knock-out mice. The dose adopted was based on the report by
Karim et al. (2001).
(RS)-DHPG injection in wild-type mice (n = 8) significantly
increased the mean durations of the pain behavior to 162.1% (233.8 ±
26.3 sec; p < 0.01) in the first phase and 151% (784.0 ±
22.3 sec; p < 0.01) in the second phase in comparison with
intrathalamic vehicle injection (144.1 ± 16.5 sec in the first phase
and 511.3 ± 20 sec in the second phase; n = 8;
Fig. 7). Although intrathecal
injection of (RS)-DHPG alone was reported to induce spontaneous
nociceptive behavior (Karim et al.,
2001), we did not observe any such effects with the intrathalamic
injection. We performed the same experiment in PLC4 knock-out mice. As
in the wild-type mice, thalamic injection of (RS)-DHPG caused the
enhancement of the mean duration of the pain behavior in the first phase to
177% (238.5 ± 26.8 sec; n = 8; p < 0.01) in
comparison with the vehicle injection (134.5 ± 15.0 sec; n =
8; Fig. 7). However, there was
no enhancement of the mean duration in the second phase (273.3 ± 20.5
sec; n = 8) by (RS)-DHPG injection in the knock-out mice
(decrease of 3% compared with the value of the vehicle injection; 280.2
± 20.0 sec; n = 8; p > 0.05;
Fig. 7). Co-injection of 0.2
µl of (RS)-DHPG (100 nmol) and Et-18-OCH3 (10 nmol) into the
thalamus reduced the mean duration of the pain behavior in the second phase to
50.5% in comparison with the (RS)-DHPG injection alone in wild-type
mice (n = 5; data not shown).
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Figure 7. Enhancement of formalin-induced nociceptive behavior in the second phase
after thalamic injection with the mGluR1/5 agonist in wild-type mice but not
in knock-out mice. The mean duration of pain behavior in the first phase
(within the first 5 min after formalin injection; open bars) are shown after
the second phase (cumulative duration of the pain behavior between 15 and 45
min; filled bars) in 10 min of pretreatment with vehicle or (RS)-DHPG
in wild-type mice (n = 8) and knock-out mice (n = 8).
*p < 0.01 compared with the vehicle injection. N.S., Not
significant.
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|
Responses of VPL neurons in wild-type and PLC4 knock-out mice
to formalin injection
PLC4 knock-out mice and PLC inhibitor-injected wild-type mice
exhibited attenuated pain behavior in the second phase in the formalin test,
implying that PLC4 may modulate responses of VPL neurons to formalin
injection. Thus, we performed single-unit recording in VPL and compared
neuronal responses between wild-type (n = 5) and PLC4 knock-out
mice (n = 5). There was no significant difference (p >
0.5) in the basal activity between the wild-type and knock-out mice. The
numbers of spikes during 1 min before the formalin injection were 33.1
± 25.9 (mean ± SD; n = 10) in the wild-type mice and
27.5 ± 17.6 (n = 13) in the knock-out mice.
Figure 8A shows an
example of the response of a VPL neuron of a wild-type mouse. After the
formalin injection, all VPL neurons in the wild-type mice showed a biphasic
response profile similar to those previously reported for the peripheral C
fiber and dorsal horn of the spinal cord (Dickenson and Sullivan
1987a,1987b;
Haley et al., 1990;
McCall et al., 1996;
Puig and Sorkin, 1996).
Immediately after the injection, the firing rate of VPL neurons increased
drastically and remained elevated for 5 min. The time course of the
response during this early phase (0-5 min) corresponded to that in the first
phase of pain behavior in the formalin test
(Fig. 1, wild-type). The firing
rate then dropped abruptly to the basal activity, followed by a quiescent
period lasting for 10 min. Fifteen minutes after the injection, the
firing rate increased again: the increase was progressive but often
interrupted by prolonged quiescent periods. This late phase (from 15 min after
the injection) continued until the end of the recording period. The saline
injection was conducted as a control experiment. The firing response increased
transiently but lasted only 17.6 ± 7.6 sec (n = 3) after the
saline injection.
In contrast to wild-type mice, the initial drastic increase in the firing
rate of a VPL neuron induced by formalin injection in PLC4 knock-out
mice was limited to the first 5 min (early phase), as shown in
Figure 8B. The firing
rate then decreased to approximately the basal activity, which was maintained
during the rest of the recording period. To compare the time courses of the
response of VPL neurons and that of the pain behavior in the early phase in
the formalin test, we first measured the duration of the period in which the
firing rate exceeded the basal activity (see Materials and Methods). In the
wild-type mice, the response of VPL neurons continued for 178.2 ± 21.4
sec (mean ± SD), whereas the response duration was 129.0 ± 43.7
sec in PLC4 knock-out mice. These two values were not significantly
different (p > 0.5; Fig.
9A), consistent with the observation that there was no
difference in the pain behavior in the first phase in the formalin test
between the wild-type and knock-out mice
(Fig. 1). However, the number
of spikes in the early phase was much smaller in the knock-out mice than in
the wild-type mice (Fig.
8A,B). Figure
9B shows the total number of spikes in the early phase
after formalin injection. The number of spikes of VPL neurons in wild-type
mice was 3038.2 ± 1208.7 (mean ± SD), whereas that in the
knock-out mice was only 901.8 ± 478.5 in the initial 5 min after the
injection. These two values were significantly different (p <
0.01).
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Figure 9. Comparison of the response of VPL neurons to formalin injection between
wild-type mice (n = 5) and PLC4 knock-out mice (n =
5). A, Total duration of the period in which the firing rate exceeded
the basal activity level (see Materials and Methods) in the early phase (0-5
min) after formalin injection. B, Total number of spikes in the early
phase (0-5 min) after formalin injection. C, Total duration of the
period in which the firing rate exceeded the basal activity in the late phase
(15-60 min). D, Total number of spikes in the late phase after
formalin injection. Each data point represents the mean ± SD.
*p < 0.01. N.S., Not significant.
|
|
To evaluate the response in the late phase, we then measured the total
duration of the response and the number of spikes in the late phase (15-60
min). The total duration in the knock-out mice (124.4 ± 108.6 sec) was
significantly reduced compared with that in the wild-type mice (890.2 ±
350.0 sec; p < 0.01; Fig.
9C). In the late phase, the number of spikes of VPL
neurons in the knock-out mice (911.2 ± 343.1) was also significantly
lower than that in the wild-type mice (7010.3 ± 2859.1;
Fig. 9D), indicating a
decreased activity of VPL neurons in the knock-out mice in the late phase.
This result is consistent with the observation that the pain behavior in the
second phase in the formalin test was attenuated in the knock-out mice
(Fig. 1), therefore
demonstrating a close temporal relationship between the pain behavior in the
formalin test and responses of VPL neurons in both wild-type and knock-out
mice.
|
Discussion
|
|---|
PLC4 is crucial for the formalin-induced inflammatory pain but
not acute pain
We have studied mice genetically lacking PLC4 in the formalin test,
which is commonly used to assess the acute pain and subsequent inflammatory
pain induced by tissue injury. We found no alteration in pain behavior in the
first phase, which represents acute pain response, in PLC4 knock-out
mice (Fig. 1). Consistent with
this result, acute pain responses examined in the hot plate and tail flick
tests were unaffected in PLC4 knock-out mice
(Fig. 2A,B). However,
the nociceptive behavior in the second phase, which represents the
inflammatory pain response, was evidently attenuated
(Fig. 1). These results
indicate that PLC4is a crucial element for the formalin-induced
inflammatory pain, whereas it does not contribute to acute pain.
Thalamic PLC4 is responsible for the inflammatory pain
PLC4, together with PLC1, is a major neuronal isoform with
distinct regional expression in the brain
(Tanaka and Kondo, 1994;
Watanabe et al., 1998). In the
lateral pain pathway, we found that PLC4 mRNA was the sole isoform of
PLCs expressed in the VPL of the thalamus, whereas a large amount of
PLC1 mRNA was co-expressed with PLC4 mRNA in the spinal cord
(Fig. 3). In the somatosensory
cortex, PLC1 was the sole isoform of PLCs in layer IV, which is
the primary recipient of thalamocortical nociceptive input
(Fig. 3). Thus, it is highly
likely that PLC4 plays a crucial role in nociceptive processing in the
VPL.
We provided several lines of evidence that thalamic PLC4 contributes
to the formalin-induced inflammatory pain. First, the intracerebroventricular
injection of a potent inhibitor of PLC attenuated nociceptive behavior in the
second phase in a dose-dependent manner in wild-type mice
(Fig. 5), implying that
PLC4 contributes to the inflammatory pain processing at the supraspinal
level. Second, the injection of PLC inhibitors into the VPL also resulted in
the reduction in the nociceptive behavior in the second phase in the formalin
test (Fig. 6). Because mRNA
expression of other types of PLC, such as PLC
and PLC
, is very
low in the thalamus (Ross et al.,
1989), the primary target of PLC inhibitors in the mouse VPL is
presumably PLC4. Thus, our results indicate that the thalamic PLC4
is involved in the formalin-induced inflammatory pain in the VPL.
The reduction in the mean duration of pain behavior in the second phase
after the intrathalamic injection of drugs was smaller than that after the
intracerebroventricular injection. The drugs through the
intracerebroventricular injection, presumably, were more diffused, not only in
the VPL but also in wider brain regions, including the medial thalamus.
Because both PLC1 and PLC4 mRNAs are expressed in the medial
thalamic nuclei that mediate the motivational-affective component of the pain,
it is possible that the diffusion after the intracerebroventricular injection
resulted in the larger reduction in the mean duration of the formalin-induced
inflammatory pain.
mGluR1 connecting to PLC4 in the VPL contributes to the
inflammatory pain
In situ hybridization and immunohistochemical studies showed the
adult mouse VPL to express a predominantly high level of mGluR1 but a very
weak level of mGluR5 (Masu et al.,
1991; Shigemoto et al.,
1992; Tanabe et al.,
1993; Shigemoto and Mizuno,
2000). Both mGluR1 and mGluR5 are known to stimulate PLCs
through activation of heterotrimeric G-proteins of the Gq/11 family
(Nakanishi, 1994;
Pin and Duvoisin, 1995). From
the results of the injection of AIDA (Fig.
6B), MPEP, or both, thalamic mGluR1 but not mGluR5 is
mainly involved in the modulation of inflammatory pain behavior in the
formalin test. Furthermore, we found that thalamic injection of a group I
mGluR agonist enhanced pain behavior in the second phase in the wild-type
mice, but this enhancement was impaired in PLC4 knock-out mice
(Fig. 7). Co-injection of PLC
inhibitor with the group I mGluR agonist into the thalamus of the wild-type
mice also impaired the enhancement of the pain behavior in the second phase.
These results indicate that mGluR1 connects to PLC4 in the VPL and that
this cascade is responsible for the formalin-induced inflammatory pain. The
thalamic mGluR1 activation somewhat enhanced the pain behavior in the first
phase in both wild-type and PLC4 knock-out mice
(Fig. 7), suggesting that the
signal cascade other than mGluR1-PLC4 signaling may be involved in the
acute pain response.
PLC4 regulates the response of VPL neurons to the inflammatory
pain
It is generally proposed that neuronal responses in the VPL to nociceptive
input account for pain behavior (Guilbaud
et al., 1990), much like in the dorsal root ganglion and spinal
dorsal horn (Hylden et al.,
1989; Young et al.,
1997). Guilbaud et al.
(1986,
1987a,1987b)
reported that responses of rat VPL neurons are enhanced after peripheral
inflammation. In the present study, the time course of the response of VPL
neurons in wild-type mice corresponded well to that of the biphasic pain
behavior in the formalin test (Figs.
1,
8A). The response of
VPL neurons in knock-out mice peaked only in the early phase and was
significantly attenuated in the late phase
(Fig. 8B), which also
correlated well with the pain behavior. The major ascending sensory afferents
to the VPL and descending corticothalamic pathways to the VPL almost certainly
use L-glutamate as the transmitter
(DeBiasi and Rustioni, 1990;
Broman and Ottersen, 1992;
DeBiasi et al., 1994). This
glutamate, acting at thalamic NMDA receptors and mGluRs, mediates nociceptive
input to thalamic neurons (Salt and Eaton,
1994,
1996;
Eaton and Salt, 1996;
Kolhekar et al., 1997). Among
mGluRs, we found that thalamic mGluR1, which connects to PLC4, is
involved in the inflammatory pain. Both mGluR1
(McCormick and von Krosigk,
1992; Golshani et al.,
1998) and PLC (Takano et
al., 1996; Lee and Boden,
1997; Lee et al.,
1999) have excitatory or depolarizing effects in various brain
tissues. Interestingly, thalamic mGluR1 shows feature localization that exists
at postsynaptic sites of the corticothalamic synapses but not at the
spinothalamic synapses (Liu et al.,
1998). On the basis of these results, we speculate that the
thalamic mGluR1-PLC4 signaling cascade at the corticothalamic synapses
provided the feedback excitation for the VPL. The feedback mechanism via this
cascade may greatly contribute to the coding for the continued pain of
formalin-induced inflammation. Reduction in the firing rate in knock-out mice
presumably resulted from blockade of the feedback excitation via this
mGluR1-PLC4 cascade.
In the first phase of pain behavior in the formalin test, the durations of
paw licking and lifting were not significantly different between the knock-out
and wild-type mice. However, spikes of VPL neurons in the early phase in
knock-out mice decreased compared with those in the wild-type mice but still
significantly exceeded the basal activity (Figs.
8,
9). Because most nociceptive
neurons in the VPL encode stimulus intensity by their firing rate
(Guilbaud et al., 1980;
Peschanski et al., 1980),
there seems to be a discrepancy between the pain behavior and the response of
VPL neurons in the knock-out mice. The exact reason for this discrepancy is as
yet unknown. This, however, may be attributable to the method by which the
pain behavior was evaluated; the total time that mice spent in licking and
lifting their paws in our study is an appropriate score for quantifying the
time course of pain behavior but is not sufficient for evaluating the
magnitude of pain. Another possibility is that the activity of other
pain-related brain regions, such as the medial thalamus, may also be involved
in the formalin-induced pain behavior, which is voluntary pain behavior.
Spinal PLC4 in the inflammatory pain
The activity of populations of spinal cord nociceptive neurons stimulated
by the hindpaw formalin injection has been assessed by expression of c-Fos
protein (Williams et al.,
1989, Todd et al.,
1994). Because of a lack of a specific inhibitor of PLC, we
counted the number Fos-LI neurons in the dorsal horn of the wild-type and
knock-out mice to estimate the contribution of spinal PLC4 during the
second phase. Fos expression, which is detected 2 hr after the formalin
injection, is maintained by neuronal activity of the dorsal horn of the spinal
cord during the second phase in the formalin test
(Abbadie et al., 1997;
Malmberg et al., 1997), and
the activity in the two phases of the formalin test contributes to the pattern
of Fos expression in the laminae of the dorsal horn in the spinal cord
differently (Abbadie et al.,
1997). In our study, the numbers of Fos-LI neurons in all laminae
of the dorsal horn of the spinal cord demonstrated no difference between
PLC4 knock-out and wild-type mice
(Fig. 4). Thus, the
contribution of spinal PLC4 is relatively minor, if any, in the second
phase of the formalin-induced pain behavior. Coderre
(1992) reported that the
intrathecal pretreatment of a PLC inhibitor reduces the formalin-induced pain
behavior in the second phase in the rodent, suggesting that other types of
spinal PLCs, such as PLC, may contribute to the inflammatory pain
sensation at the spinal level. Alternatively, spinal PLC1, which is a
counterpart of PLC4, may compensate for the lack of PLC4 function
in the dorsal horn in knock-out mice. Yashpal et al.
(2001) reported that
intrathecal treatment with an mGluR1/5 antagonist does not influence
formalin-induced activation of spinal PKC, suggesting that the spinal group I
mGluR-PLC-PKC cascade is not crucial for the formalin-induced
nociception. Furthermore, a recent study revealed the existence of
extracellular signal-related kinase, which is downstream of spinal group I
mGluRs and active in modulation of nociceptive transmission of
formalin-induced inflammatory pain at the spinal level
(Karim et al., 2001). The
signal cascade in the dorsal horn of the spinal cord could therefore be
expected to be different from the thalamic mGluR1-PLC4 cascade, even if
spinal PLC contributes to formalin-induced inflammatory pain behavior.
In conclusion, our results demonstrate that the thalamic mGluR1-PLC4
cascade is crucial for processing formalin-induced inflammatory pain. This
cascade is therefore a possible target for novel treatments of chronic
inflammatory pain at the thalamic level.
|
Footnotes
|
|---|
Received March 27, 2003;
revised June 13, 2003;
accepted June 20, 2003.
This work was supported by Grants-in-Aid for Scientific Research 13041052,
13035046, 15029254, and 15680012 from the Ministry of Education, Culture,
Sports, Science and Technology, Japan (M.M). M.M. is also grateful for the
grant from the Brain Science Foundation. We thank Drs. K. Imoto and K. Cheng
for reviewing this manuscript. We also thank Drs. Y. Yajima, M. Narita, and N.
Shibata for technical advice.
Correspondence should be addressed to Mariko Miyata, Department of
Information Physiology, National Institute for Physiological Sciences,
Myodaiji, Okazaki 444-8585, Japan.
E-mail/mmiyata{at}nips.ac.jp.
Copyright © 2003 Society for Neuroscience
0270-6474/03/238098-11$15.00/0
|
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Abstract
Introduction
