Department of Anatomy and Neurosciences and Marine Biomedical Institute, The University of Texas Medical Branch, Galveston, Texas 77555-1069
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ABSTRACT |
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Neugebauer, Volker,
Ping-Sun Chen, and
William D. Willis.
Groups II and III Metabotropic Glutamate Receptors Differentially
Modulate Brief and Prolonged Nociception in Primate STT Cells.
J. Neurophysiol. 84: 2998-3009, 2000.
The heterogeneous family of G-protein-coupled
metabotropic glutamate receptors (mGluRs) provides excitatory and
inhibitory controls of synaptic transmission and neuronal excitability
in the nervous system. Eight mGluR subtypes have been cloned and are
classified in three subgroups. Group I mGluRs can stimulate phosphoinositide hydrolysis and activate protein kinase C whereas group
II (mGluR2 and 3) and group III (mGluR4, 6, 7, and 8) mGluRs share the
ability to inhibit cAMP formation. The present study examined the roles
of groups II and III mGluRs in the processing of brief nociceptive
information and capsaicin-induced central sensitization of primate
spinothalamic tract (STT) cells in vivo. In 11 anesthetized male
monkeys (Macaca fascicularis), extracellular recordings were
made from 21 STT cells in the lumbar dorsal horn. Responses to brief
(15 s) cutaneous stimuli of innocuous (brush), marginally and
distinctly noxious (press and pinch, respectively) intensity were
recorded before, during, and after the infusion of group II and group
III mGluR agonists into the dorsal horn by microdialysis. Different
concentrations were applied for at least 20 min each (at 5 µl/min) to
obtain cumulative concentration-response relationships. Values in this
paper refer to the drug concentrations in the microdialysis fibers;
actual concentrations in the tissue are about three orders of magnitude
lower. The agonists were also applied at 10-25 min after intradermal
capsaicin injection. The group II agonists
(2S,1'S,2'S)-2-(carboxycyclopropyl)glycine (LCCG1, 1 µM-10 mM,
n = 6) and
()-2-oxa-4-aminobicyclo[3.1.0]hexane-4,6-dicarboxylate (LY379268; 1 µM-10 mM, n = 6) had no significant effects on the responses to brief cutaneous mechanical stimuli (brush, press, pinch)
or on ongoing background activity. In contrast, the group III agonist
L(+)-2-amino-4-phosphonobutyric acid (LAP4, 0.1 µM-10 mM,
n = 6) inhibited the responses to cutaneous mechanical
stimuli in a concentration-dependent manner, having a stronger effect on brush responses than on responses to press and pinch. LAP4 did not
change background discharges significantly. Intradermal injections of
capsaicin increased ongoing background activity and sensitized the STT
cells to cutaneous mechanical stimuli (ongoing activity > brush > press > pinch). When given as posttreatment, the
group II agonists LCCG1 (100 µM, n = 5) and LY379268
(100 µM, n = 6) and the group III agonist LAP4 (100 µM, n = 6) reversed the capsaicin-induced
sensitization. After washout of the agonists, the central sensitization
resumed. Our data suggest that, while activation of both group II and
group III mGluRs can reverse capsaicin-induced central sensitization,
it is the actions of group II mGluRs in particular that undergo
significant functional changes during central sensitization because
they modulate responses of sensitized STT cells but have no effect
under control conditions.
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INTRODUCTION |
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G-protein-coupled metabotropic
glutamate receptors (mGluRs) play important roles in various forms of
neuroplasticity and are becoming novel therapeutic targets for certain
neurological and psychiatric disorders associated with neuroplastic
changes (Bordi and Ugolini 1999; Conn and Pin
1997
; Knöpfel et al. 1995
;
Nicoletti et al. 1996
). The heterogeneous mGluR family
consists of eight cloned subtypes, which are classified into three
groups. Group I receptors (mGluRs 1 and 5) couple to phospholipase C
and regulate neuronal excitability and synaptic transmission. Group II
(mGluRs 2 and 3) and group III (mGluRs 4, 6, 7, and 8) receptors
inhibit adenylyl cyclase and reduce neuronal excitability and synaptic transmission (Bushell et al. 1999
; Conn and Pin
1997
; Gereau and Conn 1995
; Knöpfel
et al. 1995
; Macek et al. 1996
; Miller
1998
; Neugebauer et al. 1997
, 2000
;
Schoepp et al. 1999
; Schoppa and Westbrook
1997
; Schrader and Tasker 1997
).
An emerging field of research implicates mGluRs in nociception and
hyperalgesia. Whereas the first reports on the involvement of mGluRs in
spinal nociceptive processing relied on a relatively nonselective mGluR
antagonist (Neugebauer et al. 1994; Young et al.
1994
), recent electrophysiological and behavioral studies used
more selective agents to demonstrate a role of group I mGluRs and, in
particular, the mGluR1 subtype in prolonged nociception in the spinal
cord (Budai and Larson 1997
; Fisher and Coderre 1996
; Fundytus et al. 1998
; Neugebauer et
al. 1999
; Young et al. 1997
, 1998
).
The role of group II and group III mGluRs in the modulation of spinal
nociceptive processes is not clear yet. Activation of these receptors
has been shown to inhibit neuronal excitability and synaptic
transmission in many brain areas (see references in Conn and Pin
1997; Knöpfel et al. 1995
; Miller
1998
; Neugebauer et al. 1997
; Pin and
Duvoisin 1995
; Schoepp et al. 1999
) as well as
in spinal cord in vitro preparations (Cao et al. 1997
;
Dong and Feldman 1999
; Jane et al. 1996
;
King and Liu 1996
). With regard to nociception, an
electrophysiological study of wide-dynamic-range (WDR) spinal dorsal
horn neurons in vivo described inhibitory effects of a group II mGluR
agonist on C-fiber-evoked discharges in rats with carrageenan
inflammation, whereas mixed (excitatory or inhibitory) effects were
observed in control rats without inflammation (Stanfa and
Dickenson 1998
). A behavioral study found that the nociceptive
responses in the second phase of the formalin test were potentiated by
a group II agonist but slightly reduced by a group III agonist
(Fisher and Coderre 1996
).
The present electrophysiological study of primate spinothalamic tract
(STT) cells is the first to address the role of group II and group III
mGluRs in brief nonnociceptive and nociceptive transmission and in
prolonged nociception evoked by intradermal capsaicin. Intradermal
injection of capsaicin in humans results in primary mechanical and
thermal hyperalgesia at the injection site as well as in secondary
mechanical hyperalgesia (increased pain from noxious stimuli) and
mechanical allodynia (pain evoked by innocuous stimuli) in an area
surrounding the zone of primary hyperalgesia (LaMotte et al.
1991; Simone et al. 1991
). Primate STT neurons
develop enhanced responses to cutaneous mechanical stimuli and
increased background activity after intradermal capsaicin injection
(Dougherty and Willis 1992
; Simone et al.
1991
). Central nervous system changes, termed central
sensitization, are believed to underlie capsaicin-induced secondary
hyperalgesia and allodynia since afferent nerve fibers supplying the
area outside of primary hyperalgesia zone do not sensitize
(Baumann et al. 1992
; LaMotte et al.
1992
).
Capsaicin-induced central sensitization of STT cells involves the
activation of N-methyl-D-aspartate (NMDA) and
non-NMDA glutamate receptors, group I metabotropic glutamate receptors
of the mGluR1 subtype, and neurokinin 1 and 2 receptors
(Dougherty et al. 1992, 1994
; Neugebauer et al.
1999
; Rees et al. 1998
). Intracellular signal
transduction systems such as the protein kinase C (PKC), cAMP-dependent
protein kinase A (PKA), and the nitric-oxide-activated cGMP-dependent
protein kinase G (PKG) pathways also play important roles in the
sensitization of STT neurons after intradermal capsaicin (Lin et
al. 1996b
, 1997
, 1999
; Sluka 1997a
; Sluka
and Willis 1998
; Sluka et al. 1997
; Wu et
al. 1998
).
Activation of the G-protein-coupled group II and group III mGluRs has
been shown to inhibit cAMP formation in expression systems, brain
slices, and neuronal cultures (Conn and Pin 1997;
Pin and Duvoisin 1995
; Schoepp et al.
1999
). It is not clear, however, whether group II and/or group
III mGluRs actually couple to inhibition of neurotransmitter-induced
cAMP increases in native systems. If so, agonists at these receptors
may be useful in downregulating the enhanced responses of nociceptive
neurons in those instances of central sensitization that involve the
cAMP-PKA pathway. The functional role of group II and group III mGluRs
in spinal nociceptive processing and central sensitization was
addressed in the present study. In anesthetized monkeys, extracellular
recordings were made from STT cells. The responses to brief cutaneous
stimuli were tested before, during, and after the application of
selective group II and group III mGluR agonists into the dorsal horn by microdialysis. Cumulative concentration-responses curves were measured.
The agonists were also applied 10-25 min after intradermal injections
of capsaicin to analyze the potential usefulness of agents targeting
groups II and III mGluRs in the treatment of prolonged pain associated
with central sensitization. Preliminary results have been reported in
abstract form (Chen et al. 1998
).
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METHODS |
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Animal preparation and anesthesia
Adult male monkeys (Macaca fascicularis,
n = 11, 2.1-2.9 kg) were initially tranquilized with
ketamine (10 mg/kg im). Anesthesia was induced with a mixture of
halothane, nitrous oxide, and oxygen followed by -chloralose (60-90
mg/kg iv) and maintained by continuous intravenous infusion of
pentobarbital sodium (5 mg · kg
1
· h
1 ). After a tracheotomy, animals were
paralyzed with pancuronium (0.4-0.5 mg/h iv) and ventilated
artificially to maintain the end-tidal CO2
between 3.5 and 4.5%. A bilateral pneumothorax minimized movements
caused by respiration. Arterial oxygen saturation was monitored with a
rectal oxymeter probe and kept between 96 and 100%. Throughout the
experiment, the level of anesthesia was monitored by frequently
examining pupillary size and reflex responses and by continuously
recording CO2 levels and electrocardiogram (ECG). Core body temperature was kept at ~37°C using a thermostatically controlled heating blanket.
A laminectomy exposed the lumbar enlargement. A pool was formed with the skin flaps overlying the cord and filled with mineral oil, which was kept at 37°C using a heating device. The dura mater was opened and reflected to expose the cord. A craniotomy was performed for stereotaxic placement of a monopolar stimulating electrode into the ventroposterolateral (VPL) nucleus of the thalamus. The stereotaxic coordinates were: A: 8 mm; L: 8 mm; 16-18 mm from the cortical surface. To ensure correct placement, the thalamic electrode was initially used to record the potentials evoked by electrical stimulation of the contralateral dorsal columns and responses to cutaneous stimulation of the contralateral hind limb.
Microdialysis
For drug application, three microdialysis fibers (Spectrum Scientific, 18-kDa cutoff) were positioned in the lumbar enlargement in areas most responsive to stimulation of the lower hindlimb. The fibers were made from Cuprophan tubing (150 µm ID; wall thickness, 9 µm). The tubing was coated with a thin layer of silicone rubber (3140RTN, Dow Corning) except for a 1-mm-wide gap that was positioned in the gray matter of the ipsilateral spinal dorsal horn. Each fiber was pulled through the spinal cord just below the dorsal root entry zone using a stainless steel pin cemented in the fiber lumen. The fibers were placed in different segments of the lumbar enlargement (L5-L7), about 10 mm apart. Using polyethylene tubing, the microdialysis fibers were connected to a syringe seated in a Harvard infusion pump and were continuously perfused with artificial cerebrospinal fluid (ACSF), containing (in mM) 125.0 NaCl, 2.6 KCl, 2.5 NaH2PO4, 1.3 CaCl2, 0.9 MgCl2, 21.0 NaHCO3, and 3.5 glucose, at a rate of 5 µl/min. The ACSF was oxygenated and equilibrated to pH 7.4 with a mixture of 95% O2-5% CO2. In the beginning of each experiment, ACSF was pumped through the fibers for at least 2 h to allow the neurochemical milieu to reach equilibrium.
Administration of drugs
After stable control responses of a neuron were recorded during
administration of ACSF into the dorsal horn for 0.5-1.5 h, the
following drugs (concentrations given in parentheses) were administered
by microdialysis at 5 µl/min for 20-30 min through the fiber closest
to the recorded STT neuron (for mGluR subgroup selectivity of the
compounds, see Cartmell et al. 1999, 2000
; Conn
and Pin 1997
; Monn et al. 1999
; Schoepp
et al. 1999
): (2S,1'S,2'S)-2-(carboxycyclopropyl)glycine (LCCG1, a traditional group II mGluR agonist; 1 µM to 10 mM); (
)-2-oxa-4-aminobicyclo[3.1.0]hexane-4,6-dicarboxylate (LY379268, a
novel potent and selective group II mGluR agonist; 1 µM to 10 mM);
L(+)-2-amino-4-phosphonobutyric acid (LAP4, a potent and selective key
group III mGluR agonist; 0.1 µM to 10 mM). LCCG1 and LAP4 were
purchased from Tocris. LY379268 was a generous gift from Eli Lilly.
Stock solutions (100 mM) were prepared that were diluted in ACSF to the
indicated final concentrations on the day of the experiment. Numbers
given throughout this paper refer to the drug concentrations within the
microdialysis fiber. The amount of drug diffusing across the dialysis
membrane was estimated in vitro. These experiments have shown that in a
chamber containing 100 µl ACSF the concentration of these drugs is
about 1-3% of that being perfused at a rate of 5 µl/min for 30 min.
It is estimated that due to diffusion in the tissue the concentration
reached at the recorded neuron is another order of magnitude lower,
i.e., about 1/1,000 of the drug concentration in the microdialysis
fiber (see Benveniste and Huttemeier 1990
;
Neugebauer et al. 1999
; Sluka et al.
1997
; Sorkin et al. 1988
).
Recording of STT cells
STT neurons were recorded extracellularly in the lumbar
enlargement of the spinal cord using carbon filament electrodes
(resistance: 4 M). Cells were recorded within 0.5-1 mm of a
microdialysis fiber to ensure that the drugs administered by
microdialysis would reach the neuron in a short period of time at a
sufficient concentration. Single STT neurons were isolated following
antidromic activation from the contralateral VPL nucleus by square-wave
current pulses (1 Hz, 1 mA, 200 µs) as described previously
(Neugebauer et al. 1999
). Criteria for antidromic
activation were as follows (see Trevino et al. 1973
):
constant latency of the evoked spike; ability to follow high-frequency
(333-500 Hz) stimulation; and collision of orthodromic spikes with
antidromic spikes. The extracellularly recorded signals were amplified
and displayed on analog and digital storage oscilloscopes. Signals were
also fed into a window discriminator whose output was processed by an
interface (CED 1401) connected to a Pentium PC. Spike2 software (CED)
was used to create peristimulus rate histograms on-line and to store
and analyze digital records of single-unit activity off-line.
Throughout the experiment spike size and configuration were
continuously monitored on the digital oscilloscope and with the use of
Spike2 software to confirm that the same neuron was recorded and that
the relationship of the recording electrode to the neuron remained constant.
Experimental protocol
Once an STT cell was identified and isolated, ongoing background activity and responses to graded mechanical stimuli were recorded, and the cutaneous receptive field in the hindlimb was mapped. Cells were characterized by their responses to the following stimuli applied to the most responsive sites in the receptive field: brush (brushing the skin with a soft-hair artist's brush in a stereotyped manner), press (firm pressure using a large arterial clip to apply 1,005 g/8 mm2, which is marginally painful when applied to the skin in humans), and pinch (using a small arterial clip to apply 2,660 g/4 mm2, which is clearly painful without causing overt damage to the skin). All cells included in this study were WDR STT cells, which responded consistently to innocuous stimuli but were more strongly activated by noxious stimuli.
The stimuli were delivered at three to five test points chosen to span the receptive field. Each stimulus was applied for 15 s followed by a 15-s pause before the next test site was stimulated. To minimize a potential "human factor" bias, the experimenter who applied the mechanical stimuli did not observe the oscilloscope or the computer monitor and was unaware of the response magnitude. The entire sequence of mechanical stimuli (brush, press, and pinch) was repeated three times before drug application and before capsaicin injection, respectively. The stimuli were also applied during and after the application of different drug concentrations and after capsaicin injection. Capsaicin (0.1 ml, diluted in Tween 80 and saline at 3%) was injected intradermally between two test sites at the most responsive portion of the receptive field. Care was taken that the test stimuli were applied to areas away from the capsaicin injection site, i.e., in the zone of secondary hyperalgesia.
Data analysis
Recorded activity was analyzed off-line from peristimulus rate histograms using Spike2 software. Background activity was subtracted from the evoked responses. The effects of drugs and capsaicin on the responses to cutaneous mechanical stimuli were similar for the selected test sites (see Figs. 1, 2, and 4). Therefore the responses evoked from the different stimulation sites were averaged for each of the mechanical stimuli (brush, press, pinch). All averaged values are given as the means ± SE. Cumulative concentration-response relations were constructed by averaging the mean frequency of the responses for each drug concentration; the averages were then expressed as percentage of predrug control values (set to 100%). EC50s and the respective 95% confidence intervals were calculated from sigmoid curves fitted to the cumulative concentration-response data using the following formula for nonlinear regression (Prism 3.0, GraphPad Software, San Diego, CA): y = [A + (B - A)]/[1 + (10C/10X)D], where A = bottom plateau, B = top plateau, C = log (EC50), D = slope coefficient. Significance of the effects of LAP4 on ongoing and evoked activity was determined using a repeated-measures ANOVA to analyze the nonlinear concentration-response relationships. An F test (Prism 3.0, GraphPad Software) was used to analyze the linear regression fitted to the concentration-response data for LCCG1 and LY379268 since no successful curve fits were achieved using nonlinear regression analysis. A repeated-measures ANOVA, followed by Newman-Keuls multiple comparison test where appropriate (Prism 3.0, GraphPad Software), was performed on raw data to test for statistical significance of drug effects on capsaicin-induced changes in the responses to the mechanical stimuli and ongoing background activity. Statistical significance was accepted at the level P < 0.05.
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RESULTS |
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Sample of STT neurons
Recordings were made from 21 STT cells of the WDR type in the
lumbar enlargements (L5-L7)
of 11 monkeys. These neurons were recorded at depths of 840-1,606 µm
from the dorsal surface of the spinal cord. Based on the correlation
between recording depth and laminar position established for STT cells
in this laboratory (see Lin et al. 1999), 15 neurons
were in superficial dorsal horn (840-1,193 µm) and 6 neurons were in
the deep dorsal horn (1,388-1,606 µm). STT cells were identified by
antidromic activation from the VPL nucleus of the thalamus as described
in METHODS. The mean threshold for antidromic activation
was 449 ± 44 µA (150-740 µA); the mean latency was 6.1 ± 0.5 ms (4.0-10.0 ms). All 21 STT cells had cutaneous receptive
fields on the ipsilateral foot, including the toes in 15 cases. The
receptive fields of 11 neurons also included parts of the lower leg; 7 of these neurons had cutaneous receptive fields around the knee and on
the thigh. Ongoing activity ranged from 1.6 to 33.6 Hz (mean = 9.9 ± 2.5 Hz). Each cell was recorded within 0.5-1 mm of a
microdialysis fiber to ensure that the drugs administered by
microdialysis reached the neuron in a short period of time at a
sufficient concentration. Recordings were conducted no sooner than
3 h following insertion of the microdialysis fiber, which is
sufficient time for the stabilization of extracellular neurotransmitter
levels (Sorkin and McAdoo 1993
; Sorkin et al. 1988
).
Activation of group III mGluRs, but not group II mGluRs, inhibits the responses of STT cells to brief innocuous and noxious cutaneous mechanical stimuli
GROUP II MGLURS.
The modulation of ongoing background activity and of the responses to
brush, press, and pinch by group II mGluRs was studied using the
agonists LCCG1 (6 STT cells) and LY379268 (6 STT cells). Many previous
studies have relied on LCCG1 as a highly potent key group II mGluR
agonist with EC50 values at mGluR2 and mGluR3 in
the upper nanomolar range, although higher concentrations of LCCG1 have
now been shown to affect other mGluR subtypes as well (see Conn
and Pin 1997; Schoepp et al. 1999
). Therefore in
the present study we also tested the novel selective and highly potent group II mGluR agonist LY379268, which demonstrates
EC50 values in the low nanomolar range at mGluR2
and mGluR3 (Cartmell et al. 1999
, 2000
; Monn et
al. 1999
).
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GROUP III MGLURS.
In contrast, the selective group III mGluR agonist LAP4 (see
Conn and Pin 1997; Schoepp et al. 1999
)
inhibited the responses to graded mechanical stimuli in a
dose-dependent manner without a significant effect on the ongoing
discharges. Figure 4 shows extracellularly recorded spikes of one STT neuron in the lumbar spinal
dorsal horn (depth 1,030 µM). Ongoing activity and the responses to
graded mechanical stimulation (brush, press, pinch; see
METHODS) of selected test points in the neuron's cutaneous receptive field (see Fig. 4, inset) were recorded in the
presence of normal ACSF administered by microdialysis into the dorsal
horn (Fig. 4A). Administration of LAP4 (100 µM) by
microdialysis inhibited the responses to brush more than to pinch and
press but had no clear effect on ongoing activity (Fig. 4B).
The effects of LAP4 were reversible after washout with normal ACSF by
microdialysis (Fig. 4C).
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Group II and III mGluR agonists reverse capsaicin-induced central sensitization in STT cells
The ability of group II and III mGluRs to modulate capsaicin-induced sensitization of STT cells was studied by administering the group II agonists LCCG1 (100 µM, 15 min; n = 5) and LY379268 (100 µM, 15 min; n = 6) and the group III agonist LAP4 (100 µM, 15 min; n = 6) into the spinal dorsal horn at 10 min after intradermal capsaicin (3%) injection. Each agonist was able to reverse the enhanced responses to brush and press following capsaicin.
GROUP II MGLURS.
Figure 6 shows the typical effects of
LCCG1 (A) and LY379268 (B) on two different STT
cells. In Fig. 6A, extracellular recordings were made from
an STT neuron in the superficial dorsal horn (1,080 µM) of the lumbar
enlargement. Brief (15 s) graded mechanical stimuli (brush, press,
pinch) were applied to the skin of the neuron's receptive field.
Consistent with previous studies from this laboratory (Dougherty
and Willis 1992; Simone et al. 1991
), intradermal injection of capsaicin (3%) clearly enhanced the ongoing activity and the responses to brush and press to 252, 192, and 170%,
respectively, of the control values before capsaicin; the pinch-evoked
responses increased to only 112%. Administration of LCCG1 (100 µM;
15 min) into the dorsal horn by microdialysis reduced the enhanced
responses almost to the levels before capsaicin. The responses
increased again after washout of drug with normal ACSF and returned to
baseline at about 2 h after capsaicin. Without any pre- or
posttreatment, the capsaicin-induced sensitization of STT cells
typically lasts for about 2 h (Dougherty and Willis 1992
; Lin et al. 1999
; Simone et al.
1991
).
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GROUP III MGLURS. The group III agonist LAP4 was also able to reverse capsaicin-induced central sensitization in STT cells as shown in Fig. 8. Ongoing activity and responses to graded mechanical stimuli (brush, press, pinch) were recorded extracellularly from an STT cell in the superficial dorsal horn (840 µM) of the lumbar enlargement. Intradermal injection of capsaicin (3%) clearly enhanced ongoing background activity and the responses to brush and press to 187, 245, and 164%, respectively, of the control values before capsaicin. Pinch responses increased to only 111%. Administration of LAP4 (100 µM; 15 min) into the dorsal horn by microdialysis at 10 min post capsaicin reversed the increase in ongoing activity and reduced the enhanced brush and press responses to below the control level before capsaicin. Ongoing activity and evoked responses increased again after washout of drug with normal ACSF and showed a decline toward baseline levels at about 2 h after capsaicin.
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DISCUSSION |
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This study addressed in primate STT cells the role of groups II and III mGluRs in brief nonnociceptive and nociceptive transmission and in central sensitization evoked by intradermal capsaicin. The major findings are as follows. 1) Administration of group II mGluR agonists into the spinal dorsal horn had no significant effect on ongoing background activity or responses to brief cutaneous mechanical stimuli. 2) Intraspinal administration of a group III agonist, however, reduced the responses to innocuous and noxious mechanical stimuli without a significant effect on background activity. 3) When given as posttreatment in capsaicin-induced central sensitization, the group II mGluR agonists now inhibited the enhanced responses to brush and press and background activity. 4) Posttreatment with the group III agonist inhibited the enhanced responses to brush and press and, in some cases, pinch. These data suggest that in central sensitization of primate STT cells the functional role of group II mGluRs changes substantially, whereas the effects mediated by group III mGluRs remain largely unchanged.
The interpretation of our findings relies on the selectivity and
appropriate concentrations of the mGluR agonists we used. The group II
agonist LCCG1 has been used in many previous studies as a highly potent
agonist at mGluR2 and mGluR3 subtypes (see Conn and Pin
1997; Schoepp et al. 1999
). We tested LCCG1 so
that our results can be compared with those studies. Since higher
concentrations of LCCG1 have been shown to affect other mGluR subtypes
as well (see Schoepp et al. 1999
), we also tested the
novel potent and selective group II mGluR agonist LY379268. LY379268
has a nanomolar affinity and potency at mGluR2 and mGluR3 and shows a
more than 1,000-fold selectivity for group II mGluRs over other mGluR
subtypes (Cartmell et al. 1999
, 2000
; Monn et al.
1999
). For the study of group III mGluRs, we used LAP4, a
potent and selective key agonist for group III mGluRs (Conn and
Pin 1997
; Pin and Duvoisin 1995
; Schoepp
et al. 1999
). LCCG1 and LAP4 have been shown by others to
inhibit transmitter release in the brain when administered by
microdialysis with a time course and at concentrations similar to our
study (Cozzi et al. 1997
; Hu et al. 1999
;
Maione et al. 1998
).
Additional evidence suggests that our approach allowed the
pharmacological discrimination of groups II and III mGluRs. We used
micromolar to low millimolar drug concentrations in the
microdialysis fiber, which produce effective nanomolar to low
micromolar concentrations in the tissue at the recorded neuron (see
METHODS). Such low concentrations have been shown to be
selective in in vitro studies (Conn and Pin 1997;
Monn et al. 1999
; Schoepp et al. 1999
).
We have measured the amount of drug diffusing across the dialysis
membrane in vitro to be only 1-3% of the perfused concentration (see
METHODS). This concentration decreases by another order of
magnitude after diffusion of the drug away from the microdialysis fiber
into the dorsal horn close enough to affect the recorded STT neuron
(cf. Sluka et al. 1997
; Sorkin et al.
1988
). Furthermore both group II mGluR agonists, LCCG1 and
LY379268, had very similar effects but behaved differently than the
group III agonist LAP4 in that the group II agonists had no significant
effects under normal conditions, whereas LAP4 reduced the responses to
cutaneous mechanical stimuli.
The differential modulation of the responses of nonsensitized STT
neurons by group II and group III mGluRs is an unexpected but
intriguing finding. It is not clear why group II agonists had no effect
under normal conditions. Group II mGluR2 and mGluR3 subtypes have been
detected in the dorsal horn at the messenger RNA (Berthele et
al. 1999; Ohishi et al. 1993a
,b
; Valerio
et al. 1997
) and the protein level (Jia et al.
1999
; Tang and Sim 1999
). The cellular
localization, however, of group II mGluRs in the preterminal area
distant from the transmitter-release site rather than on the
presynaptic terminals per se (Shigemoto et al. 1997
) may
play a role in the lack of group II agonist effects we observed. It is
also possible that activation of spinal group II mGluRs produces both
inhibitory and excitatory effects with the result of a zero net effect.
Electrophysiological studies have found excitatory, inhibitory, dual,
and mixed effects of group II mGluRs on spinal motoneurons and
unidentified dorsal and ventral horn neurons (Bond and Lodge
1995
; Bond et al. 1997
; Cao et al. 1995
, 1997
; Dong and Feldman 1999
; King and Liu
1996
; Stanfa and Dickenson 1998
). Group II
mGluRs can act as auto- as well as hetero-receptors to inhibit
glutamatergic and GABAergic synaptic transmission (see Conn and
Pin 1997
; Miller 1998
; Pin and Duvoisin
1995
). STT cells receive both glutamatergic and GABAergic
inputs (Lekan and Carlton 1995
) and group II mGluRs are
present on glutamatergic and GABAergic terminals in the spinal cord
(Jia et al. 1999
). In addition, the combination of group
II mGluR-mediated postsynaptic excitation (see Conn and Pin
1997
; Miller 1998
; Pin and Duvoisin
1995
) and postsynaptic inhibition (Dutar et al.
1999
; Holmes et al. 1996
; Keele et al.
1999
; Knoflach and Kemp 1998
; Sharon et
al. 1997
) may contribute to a zero net effect of group II agonists.
It is also conceivable that the group II agonists have little effect
because the available group II receptors are under some level of
activation. The intrathecal application of a group II mGluR antagonist
has recently been reported to reduce response thresholds to noxious
mechanical stimuli in awake animals, possibly suggesting a tonic group
II mGluR component in spinal cord during normal behavior (Dolan
and Nolan 2000). In spinal cord in vitro preparations, presumed
group II mGluR antagonists potentiated synaptically evoked responses of
spinal motoneurons (Cao et al. 1997
) but not of neurons
in lamina II of the dorsal horn (Chen and Sandkühler
2000
) and had no effect on dorsal root-evoked ventral root
potentials (Jane et al. 1996
). Another explanation of
the lack of group II agonist effects involves the growing body of
evidence that mGluR-mediated signal transduction is tightly regulated
by the phosphorylation state of the receptor through various kinases
(Macek et al. 1999
; Peavy et al. 1998
;
Saugstad et al. 1998
; Schaffhauser et al.
2000
). In fact Schaffhauser et al. (2000)
have
shown that mGluR2 specifically is turned off by PKA. However, previous
studies from our lab (Sluka 1997a
; Sluka and
Willis 1997
) and work done by others (Krieger et al.
2000
; Malmberg et al. 1997
;
Ohsawa and Kamei 1999
; Wei and Roerig
1998
; Young et al. 1995
) have not produced any
evidence for a significant role of PKA or adenylyl cyclase in normal
spinal transmission.
In contrast, the group III agonist LAP4 clearly inhibited the responses
of nonsensitized STT cells. Group III mGluRs can function as
presynaptic autoreceptors, inhibit adenylyl cyclase, and modulate ion
channels (Conn and Pin 1997; Miller 1998
;
Pin and Duvoisin 1995
). Inhibition of cAMP production is
unlikely to be involved in the inhibitory effects of LAP4 on
nonsensitized STT cells because neither peripheral nor spinal blockade
of the cAMP-PKA pathway affect normal responses and nociceptive
thresholds in animals (Ahlgren and Levine 1993
;
Sluka and Willis 1997
). Further, if cAMP was involved,
group II and III agonists should have similar effects since both
subgroups couple negatively to adenylyl cyclase. A general reduction of
neuronal excitability by activation of potassium currents is also
unlikely since LAP4 inhibited only the evoked responses but not ongoing activity.
The stronger inhibition by LAP4 of the brush than the press and pinch
responses could involve L-type or both L- and N-type calcium channels.
L- and N-type calcium channel blockers reduce the responses of dorsal
horn neurons to innocuous and noxious mechanical stimuli with L-type
blockers having stronger effects on the nonnociceptive responses
(Neugebauer et al. 1996). LAP4 may also influence
transmitter release "downstream" of calcium entry (Bushell
et al. 1999
). Differences in the laminar distribution and
agonist affinity of the group III subtypes could then explain the
stronger effect of LAP4 on the brush responses: mGluR7 has a much lower
affinity for glutamate and LAP4 than mGluR4 and is localized on
nociceptive afferent terminals in the superficial dorsal horn, whereas
the laminar distribution of mGluR4 is consistent with a localization on
nonnociceptive primary afferents and spinal interneurons
(Kinoshita et al. 1998
; Li et al. 1996
,
1997
; Ohishi et al. 1995a
,b
). No other group III
subtypes have been detected in the spinal cord (Berthele et al.
1999
; Valerio et al. 1997
).
In capsaicin-induced central sensitization, both group II and III
agonists inhibited the enhanced responses of STT cells. This effect may
be related to inhibition of cAMP accumulation. Both groups of mGluRs
are characterized by their negative coupling to adenylyl cyclase (see
Conn and Pin 1997; Miller 1998
;
Pin and Duvoisin 1995
; Schoepp et al.
1999
), and blockade of the cAMP-PKA pathway can reduce
capsaicin-induced mechanical hyperalgesia and allodynia (Sluka
1997a
; Sluka and Willis 1997
) and central
sensitization (Sluka et al. 1997a
). Increased cAMP
accumulation can result from the activation of group I mGluRs,
especially mGluR1 (see also Conn and Pin 1997
), which
plays a crucial role in the capsaicin-induced central sensitization of
STT cells (Neugebauer et al. 1999
).
The dramatic change in the effects of the group II mGluR agonists
following intradermal injection of capsaicin may suggest a change in
the functional role of group II mGluRs in central sensitization.
Similarly, Stanfa and Dickenson (1998) found that the
group II agonist 1S,3S-ACPD had mixed inhibitory and
excitatory effects on electrically (C-fiber)-evoked activity in dorsal
horn neurons in control animals but predominantly inhibitory effects in
the carrageenan model. Several mechanisms may account for this change:
inhibition of the cAMP-PKA transduction cascade through group II mGluRs
in central sensitization but not under normal conditions; upregulation
of group II mGluRs in central sensitization; inhibition of
high-voltage-activated calcium channels, e.g., P/Q-type channels, which
can be inhibited by group II mGluRs (see Conn and Pin
1997
; Miller 1998
) and are, at the level of the
spinal cord, involved in central sensitization but not in normal
behavior or neurotransmission (Nebe et al. 1997
;
Sluka 1997b
); and imbalance of inhibition of excitatory
and inhibitory transmission in central sensitization, which is
characterized by an increased glutamatergic transmission combined with
a decreased GABAergic tone (Dougherty and Willis 1992
;
Dougherty et al. 1992
; Lin et al. 1996a
)
so that inhibition of excitatory transmission by group II agonists
would prevail over disinhibition.
It is important to note that capsaicin-induced central sensitization of
STT cells resumed after the group II and group III agonists were washed
out. This finding implies some form of tonic activation that attempts
to produce central sensitization and is only temporarily blocked by the
activation of group II and III mGluRs. Capsaicin-induced central
sensitization of STT cells involves a variety of neurotransmitter and
neuromodulators (Dougherty et al. 1992, 1994
;
Neugebauer et al. 1999
; Rees et al. 1998
)
and signal transduction pathways (Lin et al. 1996b
, 1997
,
1999
; Sluka 1997a
; Sluka and Willis
1998
; Sluka et al. 1997
; Wu et al.
1998
). Since manipulation of each individual component of the
sensitization process has profound effects, these elements appear to
operate in series rather than in parallel. This hypothesis is
consistent with the various well-documented interactions of ionotropic
and metabotropic glutamate receptors, glutamate and neuropeptide
receptors, metabotropic glutamate receptors, and second-messenger
systems, and among signal transduction pathways in the nervous system
(for recent reviews, see Conn and Pin 1997
;
Herrero et al. 2000
; Hobbs et al. 1999
;
Hollmann and Heinemann 1994
; Majewski et al.
1997
; Miller 1998
; Pin and Duvoisin
1995
; Ramakers et al. 1997
; Riedel 1997
; Ruth 1999
; Saria 1999
;
Sassone-Corsi 1998
; Schoepp et al. 1999
;
Tanaka and Nishizuka 1994
; Van Rossum et al.
1997
; Willis et al. 1996
; Xia and Storm
1997
).
In conclusion, both group II and III agonists inhibit capsaicin-induced
central sensitization of STT cells whereas the group III, but not group
II, mGluR agonists also affect the responses of nonsensitized neurons.
For the treatment of pain states, the ideal drug should be effective in
pain-related central sensitization but have no or minimal effects under
normal conditions. The present study suggests that group II mGluR
agonists may be among those agents. In contrast, group III agonists
(this study) and group I antagonists (Neugebauer et al.
1999) can inhibit central sensitization of STT cells but also
affect normal neurotransmission in these neurons. The significant
change of the functional role of group II mGluRs in central
sensitization suggests that mGluR2 and/or mGluR3 may be useful targets
for the development of drugs to relieve persistent pain associated with
central sensitization.
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ACKNOWLEDGMENTS |
---|
We thank K. Gondesen and G. Robak for excellent technical assistance and G. Gonzales for help with the artwork. We also thank Dr. Smriti Iyengar at Eli Lilly and Company for the generous gift of LY379268.
This work was supported by National Institutes of Health Grants NS-09743 and MH-10322.
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FOOTNOTES |
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Address for reprint requests: W. D. Willis, Jr., Dept. of Anatomy and Neurosciences and Marine Biomedical Institute, The University of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555-1069 (E-mail: wdwillis{at}utmb.edu).
Received 21 July 2000; accepted in final form 11 September 2000.
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REFERENCES |
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