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The Journal of Neuroscience, December 1, 1998, 18(23):10180-10188
Antisense Ablation of Type I Metabotropic Glutamate Receptor
mGluR1 Inhibits Spinal Nociceptive Transmission
Marie R.
Young1,
Gordon
Blackburn-Munro1,
Tracey
Dickinson1,
Melanie J.
Johnson2,
Heather
Anderson1,
Immaculate
Nakalembe1, and
Susan M.
Fleetwood-Walker1
1 Department of Preclinical Veterinary Sciences, Royal
(Dick) School of Veterinary Studies, University of Edinburgh,
Summerhall, Edinburgh EH9 1QH, United Kingdom, and
2 Medical Research Council Brain Metabolism Unit, Edinburgh
EH8 9JZ, United Kingdom
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ABSTRACT |
Electrophysiological and behavioral studies point to a role of
group I metabotropic glutamate receptors (mGluR1 and
mGluR5) in mediating spinal nociceptive responses in
rats. However, antagonists with a high degree of specificity for each
of these sites are not yet available. We, therefore, examined the
effects of antisense deletion of spinal mGluR1 expression
in assays of behavioral analgesia and of electrophysiological responses
of dorsal horn neurons. Rats treated with an mGluR1
antisense oligonucleotide reagent, delivered continuously to the
intrathecal space of the lumbar spinal cord, developed marked analgesia
as measured by an increase in the latency to tail-flick (55°C) over a
period of 4-7 d. This correlated with a selective reduction in
mGluR1, but not mGluR5, immunoreactivity in the superficial dorsal horn compared with untreated
control rats, in parallel with a significant reduction in the
proportion of neurons activated by the mGluR group I agonist 3,5-dihydroxyphenylglycine (DHPG), whereas the proportion of cells excited by the mGluR5 agonist,
trans-azetidine-2,4-dicarboxylic acid
(t-ADA) remained unaffected. In contrast, rats treated
with mGluR1 sense or mismatch probes showed none of these
changes compared with untreated, control rats. Furthermore,
multireceptive dorsal horn neurons in mGluR1
antisense-treated rats were strongly excited by innocuous stimuli to
their peripheral receptive fields, but showed severe reductions in
their sustained excitatory responses to the selective C-fiber activator
mustard oil and in responses to DHPG.
Key words:
metabotropic glutamate receptors; mGluR1; mGluR5; dorsal horn; nociception; antisense oligodeoxynucleotide probe
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INTRODUCTION |
There is a large body of evidence
supporting a role for the excitatory amino acid glutamate in mediating
sensory information at the first central synapses in the dorsal horn of
the spinal cord. Glutamate receptors in mammalian brain are classified
into two functional groups: ionotropic and metabotropic receptors
(mGluRs), (Nakanishi, 1992 ). The metabotropic group of glutamate
receptors, comprising eight receptors, is subdivided into three groups,
according to their amino acid homology, pharmacological, and signal
transduction profiles: group I (mGluRs1/5), group II
(mGluRs2/3), and group III
(mGluRs4/6/7/8) (Masu et al., 1991; Abe et al., 1992;
Nakanishi, 1992; Pin and Duvoisin, 1995). The mGluRs, which couple via
G-proteins to several signal transduction pathways, regulate neuronal
excitability in the CNS by modulating a variety of ion channels (for
review, see Saugstad et al., 1995). Many of the mGluRs have been shown to be present in the spinal cord (Shigemoto et al., 1992, 1993; Ohishi
et al., 1993, 1995; Vidnyánszky et al., 1994; Anneser et al.,
1995; Romano et al., 1995; Boxall et al., 1996; Petralia et al., 1996;
Valerio et al., 1996) where they appear to play a role in mediating
nociceptive inputs in the dorsal horn of the spinal cord (Neugebauer et
al., 1994; Young et al., 1994, 1995, 1997), as well as in the thalamus
(Eaton et al., 1993). In particular (but not exclusively) group I
mGluR1/5 receptors have been implicated in nociceptive
responses (Young et al., 1994, 1995, 1997; Fisher and Coderre, 1996a,b;
Fundytus et al., 1998), and their actions may be mediated, at least in
part, by protein kinase C (Young et al., 1995). This role appears to be
more prominent after sustained noxious stimuli, such as intraplantar
formalin and carrageenan-induced inflammation, where mGluR antagonists
prolong the latency of behavioral nociceptive responses (Fisher and
Coderre, 1996b; Young et al., 1997) or inhibit the sustained responses
of dorsal horn neurons to mustard oil (Young et al., 1997).
mGluR1/5 agonists can increase the excitability of dorsal
horn neurons (Morisset and Nagy, 1996) and facilitate responses to NMDA
and AMPA receptor activation (both of which are likely to participate
in processing sustained nociceptive inputs) (Bleakman et al., 1992;
Cerne and Randic, 1992; Bond and Lodge, 1995; Jones and Headley, 1995).
Similarly, administration of mGluR1/5 agonists appears to
interact with an NMDA (and/or AMPA) receptor-dependent mechanism to
increase nociceptive behavioral responses to intradermal formalin
injection and to noxious sensory stimuli (Coderre and Melzack, 1992;
Meller et al., 1993, 1996; Fisher and Coderre, 1996b). In both
electrophysiological and behavioral studies, agonists and antagonists
with selectivity for mGluR1/5 (as well as neutralizing
antibodies) are effective (Young et al., 1995, 1997; Fisher and Coderre
1996a,b; Fundytus et al., 1998), and some of the agents used show
partial selectivity for mGluR1 over mGluR5
(Hayashi et al., 1994; Sharp et al., 1994; Brabet et al., 1995;
Kingston et al., 1995). Nevertheless, because the available
pharmacological reagents are not wholly specific, we have taken the
alternative strategy of antisense deletion of spinal mGluR1
expression to gain an unequivocal assessment of its role in nociceptive processing.
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MATERIALS AND METHODS |
Animals and evaluation of analgesia. Adult male
Wistar rats (weight 280-450 gm, Charles River, Kent, UK) were used.
Measurements of tail-flick latency (using a modified Ugo Basile
tail-flick unit made in-house) of each rat (30-55°C to the base of
the tail) were made for 3 d before, and recommencing 4 d
after implantation of an indwelling intrathecal silicone cannula
connected to an osmotic minipump for the continuous, quantitative
administration of sense, missense, and antisense mGluR1
oligonucleotides (see below).
Oligonucleotide probes and implantation of osmotic minipump.
The 21-base antisense oligodeoxynucleotides endcapped with
phosphorothioate linkages (at the positions marked by *) were designed
according to the primary sequence of the rat mGluR1 cDNA
(Houamed et al., 1991; Masu et al., 1991). The sequence of the
mGluR1 antisense probe was:
5'-G*C*CGGACCATTGTGGCGAAG*A*-3', targeted around the translation
initiation site (nucleotides  11 to +10) and will clearly not
differentiate between splice variants in the carboxyl tail region. The
complementary mGluR1 sense probe used:
5'-T*C*TTCGCCACAATGGTCCGG*C*-3', corresponds to the reverse order
of nucleotides of the above. The mismatch probe was:
5'-T*C*CGGATCATTGGGGCGACG*A*-3'. None of the oligonucleotide probes
shows internal complementarity nor resembles any other known sequences
according to the GenBank Database. Custom synthesis, HPLC-purification,
and gel filtration were performed by Oswel DNA Service (Southampton,
UK), and probes were dissolved and aliquoted in sterile 0.9% saline,
pH 7.4, to give 0.25 µg/µl final concentration for infusion, before
being stored at 20°C until use.
For continuous infusion into the intrathecal lumbar spinal cord region,
each rat was implanted with a minipump with cannula attachment, which
was assembled the day before surgery. It consisted of an osmotic
minipump [Alza Minipump, model 2001 (Palo Alto, CA); nominal
infusion rate, 1 µl/hr) attached to two lengths of sterile cannulae:
first, to the pump, a length of ~1 cm of vinyl cannula (internal
diameter 0.76 mm, outer diameter 0.99 mm) and to this was fitted
second, a length of ~6 cm of silicone cannula (internal diameter 0.64 mm, external diameter 1.20 mm) (Degania Silicone). The pump and
cannulae were filled with one of the above solutions (or saline
control) under sterile conditions, and then the cannulae were joined to
the pump, avoiding air bubble formation, before being placed in sterile
saline at 37°C overnight.
Surgery was performed under sterile conditions, with Sagatal (Rhone
Merieux, Harlow, Essex, UK; 0.06 ml/100 gm, i.p.) anesthesia, followed
by a maintenance level of halothane (Zeneca, Cheshire, UK). The
minipump was placed intramuscularly at an interscapular site, and the
caudally directed cannula was threaded through muscle close to the
exposed region of the spinal column. A small area of muscle and
vertebral bone was cleared from two dorsal thoracic segments
(T10-T12), and the tip of the cannula was placed through a small
incision, under the dura, and eased down the dorsal spinal cord by a
premeasured distance, to within the region of the lumbar segments
L3-L6. After resection of the wound, the rat was then kept for up to
7 d, and its behavior was monitored (see above) before it was
either (1) used for an electrophysiological recording experiment or (2)
perfused and tissue taken for immunohistochemistry. A further group of
rats were assessed behaviorally for 1 week subsequent to a 7 d
infusion of antisense, to assess recovery. The position of the tip of
the cannula with respect to the level of spinal segment was ascertained
at the end of each experiment, and only data from animals with correct
cannula placement (L3-L6) were used in analysis. No animal showing
abnormal gait or paralysis during the 7 d period was included in
the study.
Electrophysiological studies. To assess the physiological
effects of the loss of receptor expression after mGluR1
antisense treatment, neuronal responsiveness to ionophoretically
applied mGluR agonists was investigated. Experiments were performed on 60 rats. Under initial halothane anesthesia, the jugular vein and
trachea were cannulated. Intravenous  -chloralose (60 mg/kg) and
urethane (1.2 mg/kg) with supplementary doses of -chloralose (10 mg/ml) were given throughout the experiment as required. Core body
temperature was maintained at 37-38°C by means of a thermostatically controlled heated blanket. Animals inspired oxygen-enriched air. The
animal was placed in a stereotaxic frame, and the thoracolumbar spinal
column was supported using three pairs of swan-necked clamps. A
laminectomy was performed at L2-L5, and agar (2% in saline at 37°C)
was injected under the most rostral vertebra and over the exposed cord
to increase mechanical stability. Above the recording region, a section
of the now solidified agar was removed, the dura was removed, and
liquid paraffin (37°C) was poured over the exposed cord.
Extracellular recordings were made from single neurons in laminae
III-V through the center barrel of a seven-barelled glass
microelectrode filled with 4 M NaCl (pH 4.0-4.5, tip
diameter 4-5 µm, DC resistance 5-8 M ). The bandwidth of the
recording amplifier was 1 Hz to 7 kHz. The following drugs were
ionophoresed from the side barrels of the electrode: group I mGluR
agonist: 3,5-dihydroxyphenylglycine (DHPG), 10 mM aqueous,
pH 4.5; mGluR5 agonist:
trans-azetidine-2,4-dicarboxylic acid (t-ADA), 10 mM aqueous, pH 8.0-8.5; and the AMPA receptor agonist:
AMPA, 10 mM aqueous, pH 8.0-8.5. All compounds were
obtained from Tocris Cookson, Bristol, UK. Retention currents of 10 nA
were used to minimize drug leakage between tests. A remaining barrel
contained 1 M NaCl, pH 4.0-4.5, for automatic current
balancing, using a Neurophore BH2 ionophoresis system (Medical Systems,
Great Neck, NY) and for current controls. The resistance of all side
barrels was 20-30 M.
Recordings were made from any multireceptive neuron encountered at a
depth from the dorsal surface corresponding to laminae I-IV, as shown
in previous studies using electrophoretic deposition of dye
(Fleetwood-Walker et al., 1988, 1993). The cutaneous receptive fields
of neurons were identified by innocuous brush stimuli and were all on
the distal hindlimb. The use of a mechanized rotating fine brush to
stimulate hair follicle (A ) afferents has been described previously
(Fleetwood-Walker et al., 1985) and was qualitatively innocuous to
human skin. Further characterization was performed using noxious
radiant heat (30-48°C, rise time 5 sec, plateau temperature for 10 sec) and noxious pinch. Approximately 90% of all the cells tested in
normal animals also showed sustained responses to mustard oil. The
responses of cells to ionophoresed agonists were then explored with
drug ejection currents being increased in a stepwise manner, every
minute in steps of 10 nA from 5 to 45 nA. The response of neurons to
the chemical algogen mustard oil (allyl isothiocyanate; Sigma, Poole,
UK; 7.5% in paraffin oil) was observed after being repeatedly applied
to the receptive field area (normally ~2 cm2)
every 5 min until sustained activation occurred.
Statistical analysis of the proportion of cells activated by agonists
in the different groups of rats (normal, antisense reagent-treated, or
sense reagent-treated rats) was performed by Mann-Whitney U test.
Immunohistochemistry. To directly monitor the loss of
receptor protein expression after 5 d of intrathecal treatment
with the mGluR1 antisense oligonucleotide probe,
immunohistochemical analysis of the lumbar spinal cord was performed.
Animals taken for immunohistochemistry were deeply anesthetized with
Sagatal (0.12 ml/100 gm, i.p.) and perfused transcardially with 0.1 M PBS (containing 3 mM sodium nitrite
and 1000 U heparin, pH 7.4) before being perfused with 4%
paraformaldehyde/0.1 M PBS. A laminectomy was then
performed, and the spinal cord was removed, together with the brain,
which were then post-fixed in the same solution for a further 4-5 hr,
before being incubated in 25% buffered sucrose overnight (4°C) and
then stored in cryoprotectant (30% ethylene glycol and 20% glycerol,
in 0.05 M PBS, pH 5.5) at 70°C. Transverse microtome
sections (52 µm) were then cut from the frozen tissue, through lumbar
segments L3-L6, and suitable sections of brain tissue were used as
positive controls for the antibodies used. Tissue sections were stored
in cryoprotectant at 20°C, until use. Sections were removed from
the cryoprotectant as required, for processing for either
mGluR1 or mGluR5 immunoreactivity. Unless otherwise stated, all solutions were made up in 0.1 M PBS,
and all incubations were performed at room temperature with gentle agitation. In all steps involving antibodies, the tissue sections were
washed twice, for 10 min each, with PBS between succeeding steps.
Sections were incubated in 1% hydrogen peroxide (30 min; Sigma) to
remove any endogenous peroxidase activity, followed by incubation in
normal goat serum (1 hr) to block nonspecific binding. They were then
incubated with polyclonal antipeptide antibodies raised to rat
mGluR1 (1180-1199) (0.25 µg/ml, 48 hr, 4°C; Chemicon,
Temecula, CA) or rat mGluR5 (1152-1171) (1 µg/ml, 48 hr,
4°C; Upstate Biotechnology, Lake Placid, NY) followed by biotinylated
goat anti-rabbit IgG antibody (1: 200 in PBS, 1 hr; Vector
Laboratories, Peterborough, UK). Sections were then incubated for 90 min with an avidin-biotin complex solution (Vectastain Elite ABC kit,
Vector Laboratories). A further wash with PBS was followed by exposure
of sections to a solution of 3,3' diaminobenzidine tetrachloride (DAB;
0.2 mg/ml; Sigma) in the presence of 3% hydrogen peroxide (1 µg/ml)
to enable visualization of the receptor protein precipitate. After a
final wash in PBS, the sections were mounted onto
poly-L-lysine-coated microscope slides, allowed to air dry
before dehydration through ascending concentrations of alcohol, and
then cleared in xylene (Sigma) and mounted in DePex mountant
(BDH). Further immunohistochemical controls consisted of replacing the
primary antibodies with nonimmune goat serum, or for preabsorbtion
controls, see below. To overcome any potential problems caused by
variable development of the DAB reaction, batches of control, sense,
mismatch, and antisense-treated spinal sections were processed
simultaneously, meaning that direct comparisons between them could be
confidently made.
As a preabsorbtion control for specificity of the mGluR1
antibody, aliquots were incubated with membranes from COS 7 cells overexpressing the rat mGluR1 receptor from a construct
in pcDNA 1 (a gift from S. Nakanishi, Kyoto University, Japan)
or with membranes from control COS 7 cells. Transfections were
performed using DEAE dextran, and cells were harvested 72 hr later
(Lutz et al., 1993). Cells were homogenized, and the crude particulate fraction was washed twice in ice-cold 20 mM HEPES-NaOH, pH
7.2, with phosphatase and peptidase inhibitors. Antibody aliquots were incubated with membranes (16 hr rolling at 4°C) before use (at twice
the usual concentration). Preincubation with normal COS 7 cell
membranes had no detectable effect, showing staining in normal dorsal
horn apparently identical to that with untreated antibody, whereas the
mGluR1-expressing membranes caused virtually complete loss
of immunostaining (see Fig. 3E).
Relative quantification of immunoreactivity was achieved using an
Improvision 1.49 image analysis package at 400×. An 80 × 30 µm
region of interest (ROI) cursor was aligned consecutively, centered on
laminae I, IIouter, IIinner, and
III and, in ventral horn, 40 µm diameter ROIs were centered over
individual motoneurons. Arbitrary gray scale units (throughout the
range 1-200) were assigned to make optimal use of the range for the
given sample set. Each section was corrected for the (low) nonspecific
background levels recorded from white matter. Individual measurements
were performed on 15 or 16 separate sections for each experimental condition.
Gel electrophoresis and immunoblotting. L3-L6 spinal cord
samples, which had been frozen at 70°C were thawed into standard Laemmli lysis buffer and denatured at 100°C for 5 min. Proteins were
separated by electrophoresis on precast, 20% polyacrylamide minigels
(Phast System; Pharmacia, Piscataway, NJ) and transferred to immobilon
(Johnson et al., 1993). After blocking with Marvel overnight at 4°C,
blots were incubated with the primary mGluR1 (1:200) or
mGluR5 (1:300) antibodies. HRP-conjugated donkey
anti-rabbit IgG (1:5000) and the Enhanced Chemiluminescence kit
(Amersham, Arlington Heights, IL) were used to visualize immunoreactive bands.
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RESULTS |
The position of the implanted intrathecal cannula was verified
4-7 d after surgery to ensure that only those animals with correct
L3-L6 placements were taken for subsequent electrophysiological recording experiments or immunohistochemistry. For the animals included
in this study, cannulae were found to lie on the dorsal surface of the
spinal cord.
Measurement of behavioral nociceptive responses
Tail-flick latency was measured for 2-3 d before, and also after,
surgical implantation of an indwelling intrathecal cannula to the
lumbar spinal cord, allowing an intermediate 3 d gap for recovery
from surgery.
After continuous infusion of the mGluR1 antisense
oligonucleotide probe, there was a marked increase in tail-flick
latency reaching a peak at 4-7 d after surgery, which was
statistically significant compared with presurgery levels
(p < 0.05; Mann-Whitney U test,
n = 9), saline-infused controls, mismatch-treated, or the sense-treated rats (Fig. 1). In the
subsequent 7 d, the tail-flick latency recovered steadily to
values approaching presurgery controls, with 36 ± 5%, 78 ± 12%, and 88 ± 4% recovery at days 10, 12, and 14 after surgery,
respectively (n = 4). The mGluR1 antisense
oligonucleotide-treated animals that were subsequently taken within
7 d after minipump and cannula implantation surgery, for either
electrophysiological studies or immunohistochemical investigation, all
displayed delayed behavioral nociceptive responses.
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Figure 1.
Effects of mGluR1 sense, mismatch, and
antisense oligonucleotide administration on behavioral nociceptive
responses (tail-flick latency, in seconds, to noxious heat applied to
the base of the tail). Statistically significant increases to
tail-flick latency are indicated by * (p < 0.05; Mann-Whitney U test). Attenuation of behavioral
nociceptive responses was observed 4-7 d after continuous infusion of
the mGluR1 antisense oligonucleotide reagent ( ) in
saline to the lumbar spinal cord segments L3-L6, compared with
saline-infused controls ( ), sense ( ), or mismatch ( )
oligonucleotide-treated rats with similarly placed intrathecal
cannulae. Values are means ± SEM (n = 6-14).
Where error bars are not apparent, they fall within the dimensions of
the symbol.
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Electrophysiological recording experiments
Responses to mGlu receptor agonists
In all experimental rats that were taken for electrophysiological
recording experiments, ionophoretic application of the mGluR agonists
DHPG or t-ADA was performed at between 5 and 45 nA, at which
every minute the ionophoretic current was increased by 10 nA increments
until activation was observed, or if none was observed by 45 nA after 2 min, then the drug application was terminated. Activation of cells
occurred within the 5 sec to 2 min after the drug had initially been
applied. Most cells were activated within 50 sec of either of the drugs
being increased to 25 nA.
In normal animals, the mGluR1/5 agonist DHPG, administered
in this regimen, caused overt activation of a majority of
multireceptive cells (56%), whereas the mGluR5 agonist
t-ADA activated a significantly smaller proportion (23%).
Table 1 illustrates this, together with
corresponding data from antisense, sense, and mismatch-infused animals.
A marked and significant reduction in the proportion of cells
responding to DHPG was seen after antisense, but not the control
reagents. The possibility that nonidentical samples of neurons may have
been recorded in control and antisense-treated animals cannot be
excluded, but the proportion of cells responding to t-ADA
was not altered by any treatment. Furthermore, ionophoretic application
of AMPA (10-40 nA) consistently caused very marked and rapid
activation of all cells tested, irrespective of whether they had been
treated with oligonucleotides or were untreated controls (data not
shown).
Sensory responses
Although dorsal horn neurons displayed vigorous responses to
motor-driven innocuous brush, which was unaffected by
mGluR1 sense, mismatch, or antisense oligonucleotide
treatments (Table 2), the same neurons
showed a greatly reduced ability to respond to noxious chemical
stimulation after the mGluR1 antisense oligonucleotide treatment.
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Table 2.
Effects of sense, mismatch, and antisense
mGluR1 oligonucleotide infusions on different sensory
responses of dorsal horn neurons
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In normal, untreated animals, neuronal activity increased shortly after
application of mustard oil to the peripheral receptive field, quickly
reaching a peak (at 20-40 sec) and then slowly declining over the next
5 min. This acute chemical nociceptive response (calculated as the mean
value over 10 sec, 20-40 sec after initial application) was not
significantly altered in sense or mismatch oligonucleotide-treated
animals compared with untreated controls, whereas responses from
antisense oligonucleotide-treated animals were markedly reduced
(p < 0.05; Mann-Whitney U test; Table 2). These observations concur with the changes in acute thermal
nociceptive responses (tail-flick) seen in the behavioral experiments.
Mechanical nociceptive responses were not investigated quantitatively
in the present study.
In normal, untreated animals (n = 8), eight of nine
cells showed a sustained and incremental excitatory response to
repeated, topical application of mustard oil to their peripheral
receptive field (up to three applications, every 5 min). The average
increase in ongoing activity of these neurons after topical mustard oil application was from 0.3 ± 0.1 Hz before mustard oil to 10.1 ± 2.6 Hz of sustained activity, measured as the mean over 10 sec taken
15-18 min after initial application (Fig.
2A). Similarly, in the
mGluR1 sense oligonucleotide-treated control animals
(n = 5), seven of eight multireceptive cells showed a
sustained excitatory response, after three topical mustard oil
applications [from 0.2 ± 1.0 Hz before mustard oil application
to 7.4 ± 1.8 Hz sustained activity measured 15-18 min after
initial application (Fig. 2B)]. Six cells recorded
from mismatch-treated animals (n = 6) (Fig. 2C) also showed marked ongoing activation as in normals
(0.1 ± 0.8 Hz before repeated mustard oil application and
7.3 ± 1.6 Hz afterward). In contrast, in rats treated with the
mGluR1 antisense oligonucleotide, which displayed greatly
attenuated behavioral nociceptive responses (n = 6),
six of six dorsal horn multireceptive cells showed no significant
sustained response to repeated topical application of mustard oil to
their peripheral receptive field (even with up to five applications);
the average increase in ongoing activity being from only 0.1 ± 0.1 Hz before mustard oil, to only 0.5 ± 1.8 Hz (Fig.
2D). The sustained mustard oil-induced firing in
antisense oligonucleotide-treated animals was significantly less than
that in control or sense-treated animals (p < 0.05 by Mann-Whitney U test). All of the cells from which
we recorded full sensory responses in normals, sense, and mismatch
animals also showed clear responses to DHPG. In the six cells from
antisense animals, from which we were able to gain adequate records of
both brush and some residual mustard oil response, we found none that responded to DHPG.
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Figure 2.
Ongoing neuronal activity records showing typical
excitatory firing responses of dorsal horn neurons to innocuous brush
stimuli (), to ionophoresis of DHPG ( ), and to noxious mustard
oil stimulation ( ). A shows untreated control,
B shows mGluR1 sense
oligonucleotide-treated, C shows mGluR1
mismatch-treated, and D shows mGluR1
antisense-treated animals. All animals were tested for tail-flick
responses before electrophysiological recording and were found to
conform to the pattern displayed in Figure 1.
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Immunohistochemistry and immunoblotting for mGluR1
and mGluR5
The specificity of antibody labeling was verified by analyzing the
distribution of mGluR1 and mGluR5
immunoreactivity in the CNS. In the cerebellar cortex, the molecular
and Purkinje layers were strongly immunoreactive for
mGluR1, whereas the granular layer was less
intensely labeled. The mGluR5 immunoreactivity in the
hippocampus was highest in the molecular layer, whereas there were many
unlabeled cell bodies in the CA1 and CA3 fields. These observations are
entirely consistent with previously published observations (Shigemoto
et al., 1992, 1993; Romano et al., 1995).
In lumbar spinal cord of control untreated animals, neural elements
strongly labeled for mGluR1 were distributed in laminae I
and II of the dorsal horn, to a lesser extent in deeper dorsal horn,
and also in the ventral horn around motoneurons (Fig.
3A,F). This pattern of immunoreactivity was unchanged in animals treated with
the mGluR1 sense or mismatch oligonucleotide probe (Fig. 3B,C). In contrast, animals treated
with the antisense oligonucleotide for mGluR1, which
had displayed attenuated behavioral nociceptive responses, showed a
marked decrease in the intensity and distribution in immunoreactive
labeling for mGluR1 in the superficial layers of the dorsal
horn (Fig. 3D). These losses were routinely observed throughout the extent of L3-L6, after cannula placement at any point
within this range, thus giving a minimal estimate of the extent of
reagent diffusion. Under the present experimental conditions, the
immunoreactivity for mGluR1 that was associated with
motoneurons in the ventral horn did not appear to be altered in the
mGluR1 antisense oligonucleotide-treated rats (Fig.
3G). Antibody specificity was demonstrated by preabsorbtion
with membranes from COS 7 cells overexpressing recombinant rat
mGluR1 (Fig. 3E). Table
3 shows quantification of the
mGluR1 immunostaining as gray scale intensity measured
across different laminae of the superficial dorsal horn, demonstrating
significant reductions in inner (and to a slightly lesser extent,
outer) lamina II of antisense-treated animals compared with the other
conditions. This loss was confirmed independently in Western blots from
polyacrylamide gel electrophoresis of spinal cord lysates (Fig.
3H). These showed a single
mGluR1-immunoreactive band at ~160-175 kDa that was
clearly depleted in samples from antisense-treated animals compared
with others. In contrast, mGluR5 immunoreactivity (which
was also concentrated in the superficial dorsal horn) showed no
differences in the pattern of staining in animals treated with sense,
mismatch, or antisense mGluR1 reagents compared with
normals (Table 3). In addition, Western blots showed the main band of
mGluR5 immunoreactivity (at ~160-180 kDa) was unaltered
by mGluR1 antisense treatment (Fig.
3H).
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Figure 3.
Effects of mGluR1 sense, mismatch, and
antisense infusion on mGluR1 and mGluR5
immunoreactivity in lumbar spinal cord.
A-D show typical representations of
mGluR1 immunoreactivity in dorsal horn in control (saline),
sense, mismatch, and antisense reagent-treated animals.
E shows the virtual lack of immunoreactivity in control
dorsal horn when the mGluR1 antibody was preabsorbed with
membranes from mGluR1-overexpressing cells.
F and G show mGluR1
immunoreactivity in ventral horn in control and mGluR1
antisense-treated animals. These results are typical of at least five
animals in each case. Scale bars, 1.0 mm. H shows
immunoblots using mGluR1 and mGluR5 antibodies
after gel electrophoresis of lysates from spinal cord segments L3-L6
of (1) control, (2) antisense, (3) sense, and (4) mismatch-treated
animals. The running positions of the molecular weight markers are
shown in kilodaltons. Results are typical of three separate
experiments.
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Table 3.
Image analysis quantification of immunohistochemistry for
mGluR1 and mGluR5 in dorsal horn of animals
treated with mGluR1 oligonucleotides
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DISCUSSION |
Various lines of evidence point to a role of mGluRs in spinal
somatosensory processing and reflex responses. It appears that mGluRs
of group I, II, and III may all have some role to play. The group I/II
mGluR agonist, 1S,3R-ACPD
(1S,3R-1-amino-1,3-cyclopentane dicarboxylic
acid) causes postsynaptic increases in the excitability of dorsal horn
neurons (Morisset and Nagy, 1996), and it has been suggested that not
only group I, but also group II, mGluRs may contribute to this effect
(Bond and Lodge, 1995). In fact, synergistic effects of group I and
group II receptor agonists on second messenger production have been
described (Schoepp et al., 1996). Group III and group II receptors at
presynaptic sites act to inhibit synaptic inputs to ventral horn
neurons (Jane et al., 1996) and may also modulate inputs to dorsal horn
neurons. Correspondingly, mGluR7 (group III) sites have
been identified in primary afferent terminals (Ohishi et al.,
1995).
Nevertheless, the present data, together with the other available
information from electrophysiological and behavioral studies, suggest
strongly that group I mGluRs play a crucial role in physiological nociceptive inputs (Neugebauer et al., 1994; Young et al., 1994, 1995,
1997; Fisher and Coderre, 1996a,b; Fundytus et al., 1998). Specifically, our pharmacological and antisense ablation data suggest
strongly that a key role is played by mGluR1. This is consistent with our previous evidence from partially selective pharmacological agents that suggested that mGluR1 may play
the predominant role (Young et al., 1995, 1997). Both the acute
responses to mustard oil (a C-fiber selective activator) and the
incremental activity resulting from its repeated application were
severely inhibited by mGluR1 ablation (Fig. 2; Table 2),
with relative preservation of responses to innocuous brush. It is not
possible from the present results to say whether mGluR1
ablation has any specific influence on the mechanism of wind-up
(increased excitability) per se, because the necessary prerequisite of
C-fiber inputs is itself abrogated by the antisense strategy. The
behavioral studies (Fig. 1) and previous reports with partially
selective mGluR1 antagonists and
mGluR1/mGluR5 antisera (Young et al.,
1997; Fisher and Coderre, 1996a; Fundytus et al., 1998) are entirely
consistent with these observations in showing that prevention of spinal
mGluR1 function leads to inhibition of behavioral
nociceptive responses.
However, mGluR1 sites are also present in ventral horn, on
or around motoneurons (Anneser et al., 1995; Alvarez et al., 1997; Boxall et al., 1998) . Thus, effects of mGluR agents on ventral root
potentials evoked by dorsal root stimulation (Boxall et al., 1996) are
likely to represent a composite of actions in dorsal and ventral horn.
Group I mGluR agonists increase ventral root potentials elicited by
ionotropic GluR agonists (Ugolini et al., 1997), whereas intracellular
recordings from motoneurons suggest that both postsynaptic facilitatory
and presynaptic inhibitory effects are brought about by group I/II
mGluR agonists (King and Liu, 1996). Presynaptic effects of group III
mGluR agonists are also prominent in motoneuron recordings (Cao et al.,
1997). It is clear, therefore, that ventral horn effects of mGluR
agents may potentially contribute to, or at least modify, the effects of mGluR manipulations in behavioral analgesia experiments. However, increased latencies or thresholds to nociceptive stimuli were measured
in behavioral responses to intrathecally applied mGluR group I
antagonists in the absence of any overt signs of motor insufficiency
(Fisher and Coderre, 1996a; Young et al., 1997). Similarly, although
mGluR1 knock-out mice display a disruption of complex
coordination behaviors that may arise from cerebellar dysfunction, they
possess well maintained muscle strength and can organize effective
goal-oriented swimming behaviors as well as normal animals (Conquet et
al., 1994). In the present study, there was no evidence for any deficit
in motor coordination, gait, or locomotor activity in the
mGluR1 intrathecal antisense-treated animals, corresponding
to the lack of change in ventral horn mGluR1 immunoreactivity (Fig. 3F,G) after
dorsally directed infusion of oligonucleotide. So, although it is not
possible to unequivocally exclude a contribution of ventral horn
effects to the behavioral results (and reflex indices of function here
not tested), it is clear that the effects of mGluR1
ablation (focused in the dorsal horn; Fig. 3, Table 3), as defined in
the electrophysiological experiments (Table 2, Fig. 2), would alone be
quite sufficient to explain the behavioral changes that we observed
(Fig. 1) and have similarly been described in mGluR1
knock-out mice (Corsi et al., 1996).
In conclusion, the present results demonstrate that the localized
antisense ablation of mGluR1 in dorsal horn (without
affecting the congener mGluR5) results in a
selective abrogation of neuronal responses to noxious stimuli (and
perhaps also sensitization) without equivalent changes in
non-nociceptive responses. Correspondingly, reflex behavioral responses
to noxious thermal stimuli are attenuated in rats with antisense
deletion of mGluR1 in lumbar dorsal horn, in the absence of
any signs of generalized motor deficit.
| |
FOOTNOTES |
Received July 8, 1998; revised Sept. 14, 1998; accepted Sept. 16, 1998.
This work was supported by Grants 046441 and 039868 from The Wellcome
Trust. We thank The Wellcome Animal Research Unit, Royal (Dick) School
of Veterinary Studies for facilities, and Colin Warwick for preparation
of some of the diagrams.
Correspondence should be addressed to Dr. S. M. Fleetwood-Walker,
Department of Preclinical Veterinary Sciences, Royal (Dick) School of
Veterinary Studies, University of Edinburgh EH9 1QH, UK.
| |
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Top
Abstract
Introduction
