Faculty of Dentistry, University of Toronto, Toronto, Ontario M5G 1G6, Canada
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ABSTRACT |
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Chiang, Chen Yu, Chun L. Kwan, James W. Hu, and Barry J. Sessle. Effects of GABA Receptor Antagonist on Trigeminal Caudalis Nociceptive Neurons in Normal and Neonatally Capsaicin-Treated Rats. J. Neurophysiol. 82: 2154-2162, 1999. We have recently demonstrated that significant increases in cutaneous mechanoreceptive field (RF) size and spontaneous activity occur in nociceptive neurons of trigeminal subnucleus caudalis (Vc, the medullary dorsal horn) of adult rats depleted of C-fiber afferents by neonatal treatment with capsaicin. These neuronal changes in capsaicin-treated (CAP) rats are suggestive of central neuroplasticity and involve N-methyl-D-aspartic acid (NMDA) receptor mechanisms. The present study examined whether the GABAA receptor antagonist bicuculline (BIC) or the GABAB receptor antagonist 2-hydroxysaclofen (SAC) can influence the RF properties and activity of Vc nociceptive neurons classified as either nociceptive-specific or wide-dynamic range in CAP adult rats or in neonatally vehicle-treated (CON) rats. C-fiber depletion was confirmed in the CAP rats by a significant decrease in plasma extravasation of Evans blue dye in a skin area receiving topical application of mustard oil, a small-fiber excitant and inflammatory irritant. As previously reported, marked increases in cutaneous RF size and spontaneous activity occurred in Vc nociceptive neurons of adult CAP rats, compared with CON rats. GABAA receptor blockade by BIC (i.t.) in CON rats produced a significant increase in spontaneous activity and in pinch RF size and tactile RF size (or appearance of a tactile area in the RF of nociceptive-specific neurons), as well as a significant lowering of the mechanical threshold and a significant enhancement of responses to pinch stimuli applied to the RF. In CAP rats, GABAA receptor blockade also produced significant changes similar to those documented in CON rats, except for a paradoxical and significant decrease in pinch RF size and no noticeable changes in responses to pinch stimuli. GABAB receptor blockade by SAC (i.t.) did not produce any significant changes in Vc nociceptive neurons in either CON or CAP rats. These results suggest that GABAA receptor-mediated inhibition may be involved in maintaining the functional expression of Vc nociceptive neuronal properties in normal conditions, and that in animals depleted of their C-fiber afferents, some features of this GABAA receptor-mediated modulation may be disrupted such that a GABAA receptor-mediated excitation is manifested.
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INTRODUCTION |
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In the adult mammalian CNS, -aminobutyric acid
(GABA) is the main inhibitory transmitter, and numerous studies have
focused on inhibition in the spinal dorsal horn mediated by either
GABAA or GABAB receptors
(for review, see Bormann 1988
; Costa 1998
; Hammond and Graham 1997
; Sieghart
1995
). The activation of GABAA receptors
usually hyperpolarizes postsynaptic neurons by opening anion channels
and allowing an influx of chloride ions; bicuculline (BIC) can block
this effect (Bormann 1988
; Costa 1998
; Sieghart 1995
). Recent studies have shown that
GABAB receptor mechanisms are also involved in
dorsal horn modulatory mechanisms (Hammond and Graham
1997
; Hao et al. 1994
), and their agonists may
postsynaptically activate K+ channels or
presynaptically reduce the duration of
Ca2+-dependent action potentials, thus reducing
the release of transmitters from terminals in the dorsal horn
(Bormann 1988
; Grudt and Henderson 1998
;
Hammond and Graham 1997
; Kangrga et al.
1991
). These GABAergic inhibitory mechanisms have been
documented in models of inflammatory and neuropathic pain
(Castro-Lopes et al. 1994
; Hammond and Graham 1997
; Sluka et al. 1994
;
Wiesenfeld-Hallin et al. 1997
; Willis et al.
1996
). Recent studies have in addition revealed interactions between GABA and N-methyl-D-aspartate (NMDA)
receptor mechanisms. For example, activation of
GABAA receptors may enhance NMDA
receptor-mediated neuronal excitotoxicity if a positive shift in the
Cl
equilibrium potential occurs, e.g., in
cerebral ischemia (for review, see Ben-Ari et al. 1997
; Cherubini et
al. 1991
; Costa 1998
; Sieghart 1995
), and GABAA
receptor activation can also induce depolarization and promote the
expression of NMDA receptor-mediated activity in developing neurons
(see Cherubini et al. 1991
; Leinekugel et al.
1997
).
A GABAergic modulatory substrate also exists in the medullary dorsal
horn, i.e., trigeminal subnucleus caudalis (Vc) (Almond et al.
1996; Ginestal and Matute 1993
; Grudt and
Henderson 1998
; Iliakis et al. 1996
;
Kondo et al. 1995
; Matthews et al. 1989
), and in addition NMDA mechanisms have been shown to contribute to
neuroplastic changes in the mechanoreceptive field (RF) properties of
Vc nociceptive neurons and associated responses induced by nociceptive
afferent inputs in intact rats (Chiang et al. 1998
; Yu et al. 1996
). Furthermore, we have recently
demonstrated that NMDA-dependent neuroplastic changes in the RF
properties of Vc nociceptive neurons in adult rats depleted of C-fiber
afferents by neonatal treatment with capsaicin (CAP rats)
(Chiang et al. 1997
). Therefore the aim of the present
study was to test whether the GABAA receptor
antagonist BIC or the GABAB receptor antagonist 2-hydroxysaclofen (SAC) can influence the RF properties and activity of
Vc nociceptive neurons in CAP rats. Because intact rats served as a
control group, an additional aim was to test whether GABA mechanisms
serve to modulate the functional expression of Vc nociceptive neuronal
characteristics in these rats. Preliminary data have been presented in
abstract form (Chiang et al. 1996
).
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METHODS |
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Capsaicin treatment and verification of C-fiber depletion
This study was carried out in 29 male Sprague-Dawley rats.
Fifteen rats were neonatally treated with capsaicin (50 mg/kg sc, at
postnatal day 1-2, CAP rats) (for details see Gamse
et al. 1980; Kwan et al. 1996
; Ren et al.
1994
), and the remainder were vehicle-treated (CON rats); both
groups were studied electrophysiologically 2-4 mo later. We have
previously provided electronmicroscopic evidence (Kwan et al.
1999
) and spectrophotometric evidence (Chiang et al.
1997
; Kwan et al. 1996
) of marked C-fiber
depletion in CAP rats. In the present study, the spectrophotometric
method was again used to assess the effectiveness of neonatal capsaicin treatment in markedly depleting C-fiber afferents. Inflammation induced
by the cutaneous application of 0.02 ml 20% mustard oil, a small-fiber
excitant and inflammatory irritant (MO, allyl-isothiocyanate, BDH,
Toronto; diluted in mineral oil), was assessed by the plasma extravasation of Evans blue dye (EB) bound to plasma protein. The EB
solution was injected (20 mg/kg iv) into the CAP or CON adult rats at
the end of the neuronal recording experiment, followed by application
of MO to the shaved skin (diameter 1.5 cm) of the leg. Twenty minutes
later, this skin and the analogous skin area of the contralateral leg
were removed, and the amount of extravasated EB was determined
spectrophotometrically (Gamse et al. 1980
; Kwan et al. 1996
).
Animal preparation and electrophysiological experimental procedures
The methods used for animal preparation and anesthesia,
stimulation, and neuronal recording and classification were similar to
those detailed previously (Chiang et al. 1997,
1998
; Kwan et al. 1996
; Yu et al.
1993
). Briefly, rats were anesthetized with urethan (1 g/kg
ip)/
-chloralose (50 mg/kg ip), immobilized with gallamine
triethiodide, and artificially ventilated. Percentage expired
CO2, heart rate, and rectal temperature were
maintained at 3.5-4.5%, 330-420 beats/min, and 37.0-37.5°C,
respectively. Single neuronal activity was recorded extracellularly
from histologically confirmed sites in Vc. A wide range of graded
mechanical stimuli as well as noxious radiant heat stimulation
(51-53°C) were used to classify units according to previously
outlined criteria (Chiang et al. 1997
) into wide-dynamic
range (WDR) or nociceptive-specific (NS) neurons (see
RESULTS), or low-threshold mechanoreceptive (LTM) neurons
or primary afferents (not studied further).
Cutaneous tactile and pinch RFs of Vc nociceptive neurons were
determined through the use of a camel hair brush or a blunt probe and a
modified forceps (with an attached strain gauge: 10-200 g),
respectively. The RF areas were measured by a computer-aided device
(SigmaScan, Jandel, CA) (Yu et al. 1993). The background activity of each Vc nociceptive neuron was regularly recorded for a
2-min period before each RF size determination and termed spontaneous
activity (spikes/2 min). The mechanical threshold of the unit was
determined with the use of a set of von Frey nylon monofilaments
(0.1-92 g) applied to the center of the neuronal RF. The threshold was
defined as the monofilament with the lowest value that elicited 1-2
spikes/trial in at least five of six trials. Neuronal responses to
pinch stimuli were quantified as the number of spikes produced by a
sustained pinch with 200 g for 3 s.
Drug administration
BIC (bicuculline methiodide, RBI, Natick, MA), a selective
GABAA receptor antagonist (Costa
1998; Sieghart 1995
), was applied (3 µg/10
µl in saline freshly prepared, equivalent to 6.8 nmole i.t.) to the
surface of the caudal medulla overlying Vc in both CON and CAP groups.
We selected the 3-µg dose because in preliminary experiments, we
observed that 5 µg BIC i.t. application always produced nociceptive
neuronal seizurelike activity for >5 min, which would have prevented
us from observing the drug effects at the postdrug 5-min time point
(see RESULTS). The application of 1 µg BIC did
not produce any such effect, but 3 µg usually produced seizurelike
activity that ceased within 3 min; this dose is also the same dose (on
molar basis) shown to be effective in a previous study
(Sivilotti and Woolf 1994
) in influencing nociceptive neuronal RF properties. SAC (2-hydroxysaclofen, RBI, Natick, MA), a
selective GABAB receptor antagonist (Kerr et al.
1988
), was applied (4 µg/10 µl in dilute aqueous base,
equivalent to 15 nmole i.t.) to Vc in both CON and CAP rats. We chose
the dose of 4 µg, which is equimolar to that of BIC, for comparison
purposes. After a supplementary dose of urethan (220-250 mg/kg iv),
the RF properties of each neuron were tested before and after
antagonist or vehicle (saline) application: 5 min before drug
application, and thereafter at 5-min intervals for the first 10 min,
and at 10-min intervals for the subsequent 50 min observation period.
Only one single unit was recorded and analyzed in this manner in each experiment.
Histological and statistical analyses
Recording sites were marked by electrolytic (anodal current, 8 µA for 10 s) lesions and verified with conventional histological procedures.
All data values are expressed as means ± SE. For statistical comparison of values between different time points, a one-way repeated measures ANOVA or Friedman repeated measures ANOVA on ranks was used, followed by multiple comparisons with Student-Newman-Keuls method; for comparison of values between two groups of animals, either a two-way ANOVA followed by the multiple comparisons, Kruskal-Wallis ANOVA on ranks, or Mann-Whitney rank sum test was used. For comparison of amount of extravasated EB induced by cutaneous application of MO between CAP and CON rats, the Student's t-test was used. P < 0.05 was considered statistically significant.
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RESULTS |
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A total of 29 Vc neurons was functionally identified as nociceptive neurons in both CON and CAP rats: 11 as WDR neurons (CON, n = 5; CAP, n = 6), 18 as NS neurons (CON, n = 9; CAP, n = 9). All of these neurons were histologically retrieved in Vc laminae IV-VI, except for 1 neuron in laminae I-II (Fig. 1).
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Verification of C-fiber depletion in CAP rats
The amount of extravasated EB induced by application of MO to the ipsilateral (left) hindlimb skin of CAP rats (1.28 ± 0.16 µg, mean ± SE, n = 15) was significantly less (P < 0.0001, t-test) than that of CON rats (7.38 ± 1.09 µg, n = 14). The EB amount in skin samples from the contralateral hindlimb that received no application of MO was not significantly different between CAP (0.85 ± 0.09 µg) and CON (1.24 ± 0.12 µg) rats.
Effects of BIC on Vc nociceptive neuronal properties in CON and CAP rats
SPONTANEOUS ACTIVITY.
Spontaneous activity was studied in six NS and three WDR neurons in CAP
rats and six NS and two WDR neurons in CON rats. In resting conditions,
two of six NS neurons tested in CAP rats had spontaneous activity
between 9 and 231 spikes/2 min, and 4 of 6 NS neurons tested in CON
rats had spontaneous activity ranging between 1 and 3 spikes/2 min.
Consistent with our earlier findings (Chiang et al.
1997), the mean spontaneous activity of NS neurons (40 ± 38 spikes/2 min, n = 6) in CAP rats was markedly higher than that of CON rats (11.2 ± 0.5 spikes/2 min, n = 6). After BIC application, five of six NS neurons in CAP rats and all
six NS neurons in CON rats showed a significant increase (4-6 times initial activity) in their spontaneous activity, which peaked 5 min
after drug application and recovered 20-30 min later
(P < 0.05; Fig. 2 and
Table 1). Significant differences in
magnitude of the increased activity following BIC application were
found between CAP and CON rats (P < 0.05). There was
no indication that this increased activity was related to the location
(laminae V-VI) of the NS neurons tested. The spontaneous firing rate
of WDR neurons tested (3 in CAP rats and 2 in CON rats) in resting
conditions varied from 0 to 56 spikes/2 min. After BIC application, all
WDR neurons increased their firing rate (range 4-5,665 spikes/2 min) and, like NS neurons, recovered within 20-30 min. Due to the limited number of WDR neurons tested and their wide individual differences, a
statistical comparison of spontaneous activity to drug-induced activity
in CAP and CON rats could not be made.
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RF SIZE.
Also consistent with our earlier findings (Chiang et al.
1997), the mean pinch RF size of NS neurons in CAP rats was
significantly larger than that in CON rats (5.67 ± 1.52 cm2 vs. 2.77 ± 0.91 cm2, respectively,
n = 6 in each group; P < 0.05, Mann-Whitney rank sum test). Following BIC application, the pinch RF
size of NS neurons in CON rats significantly increased to four times
that of control values at 5 min (P < 0.001); in
contrast, the pinch RF size of NS neurons in CAP rats was paradoxically
and significantly reduced to 50% of control values between 5 and 10 min (P = 0.02). There was a significant difference in
the values at 5 min between these two groups (P < 0.05). The reduced pinch RF size of CAP rats after BIC
application approximated that of the control (predrug) RF size of CON
rats, and the time courses of the pinch RF changes after BIC
application in the two groups of rats were mirror images (Fig.
3; Table 1). Three WDR neurons in CAP
rats also showed abrupt and dramatic reductions in their pinch RF size to 17-33% of control values after BIC application, whereas two WDR
neurons in CON rats showed an increase in pinch RF size to 277-355%
of control values.
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MECHANICAL THRESHOLD. The mean mechanical threshold of the six NS neurons in CAP rats was comparable with that of the six NS neurons in CON rats in resting conditions (Table 1). After BIC application, the thresholds of all NS neurons in both groups were significantly decreased within 5 min (P < 0.001) to 1-7% of control values; this effect lasted for 20 min in CON rats and >30 min in CAP rats. Furthermore, the differences in threshold changes at each time point during the 60-min observation period between CAP and CON groups were significant (P < 0.05; Fig. 4B and Table 1). The threshold of four WDR neurons tested in both group of rats also dramatically decreased to 6-46% of control values, but no significant differences in the threshold changes occurred between these two groups (P > 0.3).
PINCH RESPONSE. In resting conditions, the mean pinch responses of NS neurons of both CAP and CON rats were comparable (Table 1). Soon after BIC application, the NS neuronal responses to pinch stimuli in CON rats (n = 6) were significantly increased five- to sixfold (P < 0.05) and then recovered to control values within 20 min, whereas those in CAP rats (n = 6) remained unchanged throughout the 60-min observation period. The differences at 5 and 10 min time points between CAP and CON rats were significant (P < 0.01; see Fig. 5 and Table 1). After BIC application, the pinch responses of the three WDR neurons in CAP rats changed to 26-237% of control values (increase in 2 neurons, decrease in 1 neuron), whereas those of the two WDR neurons in CON rats increased to 203-415% of control values.
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Effects of SAC on Vc nociceptive neuronal properties in CON and CAP rats
Six nociceptive (3 NS, 3 WDR) neurons in CAP rats and six nociceptive (3 NS, 3 WDR) neurons in CON rats were tested for effects of the GABAB antagonist SAC on neuronal spontaneous activity and RF properties. The experimental paradigm was identical to that used in BIC application experiments. As shown in Fig. 6, A and C, and Table 1, before SAC application the spontaneous activity and pinch RF size of these nociceptive neurons in CAP rats were markedly increased, compared with those in CON rats. No significant changes in either spontaneous activity or pinch RF size occurred during the 60-min observation period following SAC application in either CON and CAP rats. There was also no appearance of a novel tactile RF area in NS neurons and no noticeable changes in tactile RF size of WDR neurons following SAC application in either CON and CAP rats (Fig. 6B). Similarly, no significant changes in mechanical threshold or magnitude of pinch responses were found in either CON and CAP rats following SAC application (Fig. 6, D and E, and Table 1).
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DISCUSSION |
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The dosage of capsaicin that we used (50 mg/kg) is generally
accepted to be effective for depleting C-fiber afferents when administered at postnatal day 1-2 (Hammond and Ruda
1991; Ren et al. 1994
; for review, see
Buck and Burks 1986
; Fitzgerald 1983
; Holzer 1991
). We verified the effectiveness of neonatal
capsaicin treatment by documenting the marked and statistically
significant reductions in MO-induced plasma extravasation in CAP rats
(Chiang et al. 1997
; Kwan et al. 1996
;
Yu et al. 1996
); we have, in addition, documented in a
recent electronmicroscopic analysis that these rats also show a marked
depletion of C-fiber afferents (Kwan et al. 1999
).
This study has for the first time shown in the Vc that the
GABAA receptor antagonist BIC (i.t.) produces a
significant increase in spontaneous activity and in pinch RF size and
tactile RF size (or appearance of a tactile area in the RF of NS
neurons), as well as a significant lowering of the mechanical threshold
and a significant enhancement of responses to pinch stimuli applied to
the RF in both NS and WDR neurons of intact rats. These findings are
consistent with previous studies in spinal nociceptive neurons in
normal rats (Sivilotti and Woolf 1994), and with recent
findings of GABA-immunoreactive neurons in Vc and spinal dorsal horn
(Almond et al. 1996
; Alvarez et al. 1992
,
1993
; Ginestal and Matute 1993
; Iliakis et al. 1996
; Kondo et al. 1995
;
Matthews et al. 1989
). Our findings suggest that a tonic
activation of the GABAA receptor may be involved
in maintaining the functional expression of Vc nociceptive neuronal
properties in normal conditions. Furthermore, in CAP rats, application
of BIC also produced significant changes in spontaneous activity,
mechanical threshold, and the appearance of a novel tactile RF area
that were identical to those in CON rats. However, the pinch RF size of
all NS and most WDR neurons in CAP rats paradoxically and significantly
decreased abruptly from its preexisting enlarged size to a level
comparable with the normal RF size of NS and WDR neurons in CON rats.
In parallel with these RF size changes, the neuronal responses to pinch
stimuli in CAP rats, in contrast to CON rats, remained unchanged
throughout the 60-min observation period. Because all these changes in
neuronal activity and RF properties following BIC application showed a very similar transient and reversible time course and there was a lack
of significant changes in neuronal activity and response properties
following application of the GABAB antagonist SAC
in both CON and CAP rats, our findings suggest a unique
GABAA antagonist effect of BIC in both CON and
CAP rats.
In view of the structural and functional similarity between the spinal
dorsal horn and Vc, the medullary dorsal horn (see Gobel et al.
1981; Sessle 1996
), the appearance of a novel
tactile RF and a dramatic lowering of mechanical threshold and an
increase in spontaneous activity in Vc NS or WDR neurons following BIC application in intact animals may, by extrapolation, be explained by
GABAergic disinhibition, which has been documented in the spinal dorsal
horn (Sivilotti and Woolf 1994
). It has also been
demonstrated that a tactile-evoked allodynia in rats can be produced by
spinal (i.t.) BIC or strychnine (Hammond and Graham
1997
; Yaksh 1989
). Furthermore, anatomic
findings have shown that GABA-containing cells synapse onto the
terminals of low-threshold myelinated primary afferents, and in turn
receive synaptic input from low-threshold myelinated primary afferents
(Alvarez et al. 1992
; Barber et al. 1978
;
Iliakis et al. 1996
); GABA-containing cells also synapse onto spinothalamic cells (Carlton et al. 1992
;
Kondo et al. 1995
). Preexisting A-fiber inputs to
nociceptive neurons are thought to be functionally "masked" by
GABAergic inhibitory mechanisms that are driven by descending
influences and segmental afferent inputs under normal conditions
(Dickenson et al. 1997
; Sivilotti and Woolf
1994
). It is thus possible that the application of BIC in CAP
rats would block this GABAergic inhibition and thereby "unmask"
A-fiber nonnociceptive afferent inputs that can then excite nociceptive
neurons. This may especially be the case if this disinhibition were to
take place only at the A-fiber afferent terminal region (mostly in
lamina III/IV of dorsal horn) (Powell and Todd 1992
),
which is not accessed by C-fibers and which as a consequence is
unlikely to show degenerative changes following neonatal capsaicin
treatment (Nagy and Hunt 1983
; Shortland et al.
1990
). It is noteworthy that BIC-induced changes in neuronal spontaneous activity, RF size, and response properties in CAP rats were
significantly less than those in CON rats (see Table 1); this may be
related to the marked reduction in GABA receptors that has been
reported in spinal dorsal horn after neonatal capsaicin treatment
(Castro-Lopes et al. 1994
; Singer and Placheta
1980
).
In contrast to CON rats, BIC did not produce significant changes in the
magnitude of the pinch-evoked responses in CAP rats. It is possible
that two kinds of afferents may be responsible for the pinch-evoked
response: capsaicin-insensitive small-fiber afferents, which might not
be under GABAergic modulation, and capsaicin-sensitive small-fiber
afferents, which have been shown to be subject to GABAergic presynaptic
inhibition in normal conditions (Castro-Lopes et al.
1994; Singer and Placheta 1980
). In CON rats, BIC may facilitate neuronal responses to pinch stimuli due to disinhibition of these capsaicin-sensitive afferents. In CAP rats, however, BIC may not affect pinch-evoked responses, because
capsaicin-sensitive, GABA-modulated small-fiber afferents are mostly
depleted (Castro-Lopes et al. 1994
; Singer and
Placheta 1980
).
Recent findings have revealed that NMDA receptor subunits (NMDAR1) and
GABA-immunoreactive neurons are found in the spinal and medullary
dorsal horns and sensory ganglia (Almond et al. 1996;
Ginestal and Matute 1993
; Iliakis et al.
1996
; Kondo et al. 1994
, 1995
;
Matthews et al. 1989
; Petralia et al.
1994
; Shigemoto et al. 1992
). Furthermore,
generation of GABA receptors precedes that of glutamate receptors in
the developing brain (Chen et al. 1995
), GABAergic
neurons are highly susceptible to glutamate in developing neurons (van
den Pol et al. 1998
) and to excitatory amino acid-mediated
neurotoxicity (Sloper et al. 1986
), and GABA receptors
regulate substance P release from primary afferents (Liu et al. 1997
), some of which also contain glutamate
(De Biasi and Rustioni 1988
). Interestingly, numerous in
vitro electrophysiological studies have shown that a
GABAA receptor-mediated excitation occurs in
neonatal hippocampus, neocortex, hypothalamus, and spinal cord (for
review, see Ben-Ari et al. 1997
; Cherubini et al.
1991
; Sieghart 1995
) as well as in mature and
ischemia-insulted neurons (Avoli 1992
; Fukuda et
al. 1998
; Katchman et al. 1994
; Thompson
et al. 1988
; van den Pol et al. 1996
), and close
interactions may exist between GABAA, NMDA, and
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors
mainly through GABAA receptor-mediated
excitation (Gao et al. 1998
; Obrietan and van den
Pol 1995
; for review see Ben-Ari et al. 1997
).
This GABAA receptor-mediated excitation is
caused by a postsynaptic membrane depolarization resulting from a shift
of the Cl
equilibrium potential to a more
positive level (Costa 1998
; Fukuda et al.
1998
; Sieghart 1995
; Thompson et al.
1988
). Both GABAA receptor-mediated
excitation and inhibition can be blocked by BIC or picrotoxin
(Avoli 1992
; Cherubini et al. 1991
;
Sieghart 1995
). Because neonatal capsaicin treatment
causes a marked loss of C-fibers (Buck and Burks 1986
;
Fitzgerald 1983
; Hammond and Ruda 1991
;
Holzer 1991
; Ren et al. 1994
) and
structural changes and reorganization (Castro-Lopes et al. 1994
;
Shortland et al. 1990
; Singer and Placheta 1980
) in the spinal dorsal
horn, we speculate that these long-term changes may disturb the local
chemical environment and intracellular Cl
homeostasis and lead to sustained intracellular
Cl
accumulation, which gives rise to a positive
shift of ECl
and
GABAA-mediated depolarization. Although this
would require different techniques than ours to test, we believe that
this depolarization may activate voltage-dependent
Ca2+ channels and reduce voltage-dependent
Mg2+ block of NMDA receptor
channels, which eventually cause an increase in intracellular
Ca2+ concentration and neuroplastic changes
(Ben-Ari et al. 1997
; Kaila et al. 1997
;
Kakazu et al. 1999
; Leinekugel et al.
1997
; Luhmann and Prince 1990
; Thompson
et al. 1988
).
In view of these data and our previous findings that the enlarged pinch
RF of Vc nociceptive neurons in CAP rats involves a sustained NMDA
receptor activation (Chiang et al. 1997), it is
conceivable that local blockade of GABAA
receptor-mediated excitation by BIC application to Vc in CAP rats
could deactivate such a sustained NMDA receptor activation
(Ben-Ari et al. 1997
) and thus restore the normal size
of the pinch RF. Another interpretation is that the effect of BIC
application to the Vc neurons may involve structures other than Vc as a
result of the diffusion of BIC in the brain stem. This stems from
findings that the neurons in the nucleus raphe magnus and
periaqueductal gray are subject to a tonic inhibitory input from
GABAergic neurons (for review, see Fields and Basbaum
1994
) and that BIC injected into raphe magnus or the cisterna
produces antinociception (Hammond and Graham 1997
; Ueda et al. 1987
). However, following local (Vc)
application of BIC in CON or CAP rats we have observed enhancement of
pinch or tactile RF and responses that would not seem indicative of
antinociception; this suggests that the BIC did not spread to involve
these structures. It is noteworthy that N-methyl derivatives
of BIC may block the low-threshold T-type Ca2+
current-induced afterhyperpolarization
(IAHP), and that only the methyl
radical, not BIC itself, is responsible for this blockade in in vitro
conditions (Debarbieux et al. 1998
). We believe that the
effects of BIC observed in our in vivo experiments are mainly due to
the antagonism of GABAA receptors, because we
observed seizurelike activity following BIC application, which is a
characteristic of blockade of GABAergic inhibition. However, our data
do not rule out the possibility that BIC may produce other cellular
effects, such as the IAHP block, in
addition to its blockade of GABAA receptors.
GABAB receptors are pharmacologically distinct
from GABAA receptors. In dorsal root ganglion
cells, GABAB receptor activation reduces the
duration of Ca2+-dependent action potentials and
thus the release of neurotransmitters, while in CNS cells, its
activation leads to an outward K+ current that is
inhibitory (Bormann 1988). Because the application of
SAC, which is a potent antagonist of the GABAB
receptor (Kerr et al. 1988
), had no noticeable action on
Vc nociceptive neurons in both CON and CAP rats in the present study,
it seems likely that GABAB receptor mechanisms
are not involved in modulation of Vc nociceptive mechanisms, although
GABAB receptor antagonists (SAC and CGP 35348)
have been demonstrated to inhibit baclofen (GABAB
receptor agonist)-induced depression of excitatory transmission in LTM
neurons of trigeminal subnucleus oralis (Fromm et al.
1992
). Previous studies using another
GABAB receptor agonist CGP 35348 have nonetheless
shown that GABAB receptor mechanisms are involved in nociception in the spinal somatosensory system (Hammond and Washington 1993
; Hao et al. 1994
;
Wiesenfeld-Hallin et al. 1997
; Xu et al.
1993
). The discrepancy in findings may be due to differences in
the animal preparations, stimuli, or drugs used. Because the pharmacological potency of CGP 35348 is comparable to that of SAC
(Hills et al. 1991
), the lack of a SAC effect might
conceivably be due to the low dose used in our study. Nevertheless, our
findings are supported by Sluka et al. (1994)
, who have
shown that the inflammation-induced central release of excitatory amino
acids is prevented by a GABAA receptor antagonist
but not by a GABAB receptor antagonist.
Taken together, these findings have revealed that a
GABAA receptor-mediated inhibition may be
involved in maintaining the functional expression of Vc nociceptive
neuronal properties in control rats and is moderately depressed in rats
treated neonatally with capsaicin that selectively depletes C-fiber
afferents and reduces GABA receptors in the dorsal horn. We have
previously shown that NMDA receptor activation is involved in the Vc
nociceptive neuroplastic changes in CAP rats (Chiang et al.
1997), and the present study further suggests that the
sustained NMDA receptor activation in CAP rats may involve a
GABAA receptor-mediated excitation.
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ACKNOWLEDGMENTS |
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The authors thank K. MacLeod for technical assistance and Drs. Brian Cairns and Liang Zhang for comments on the manuscript.
This work was supported by National Institute of Dental and Craniofacial Research Grant DE-04786 to B. J. Sessle.
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FOOTNOTES |
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Address for reprint requests: B. J. Sessle, Faculty of Dentistry, University of Toronto, 124 Edward St., Toronto, Ontario M5G 1G6, Canada.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 11 March 1999; accepted in final form 28 June 1999.
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REFERENCES |
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