Effects of GABA Receptor Antagonist on Trigeminal Caudalis Nociceptive Neurons in Normal and Neonatally Capsaicin-Treated Rats

Chen Yu Chiang, Chun L. Kwan, James W. Hu, and Barry J. Sessle

Faculty of Dentistry, University of Toronto, Toronto, Ontario M5G 1G6, Canada


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In the adult mammalian CNS, gamma -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).


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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)/alpha -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.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 1. Recording sites plotted on a transverse section of rostral part of caudal medulla, 1.4-1.6 mm posterior to obex. Recording sites in neonatally vehicle-treated (CON) and capsaicin-treated (CAP) rats are separately displayed on the left and right side of the diagram, respectively, although they are all located ipsilateral to the stimulation (right) side.  and open circle , nociceptive-specific (NS) and wide-dynamic range (WDR) neurons, respectively, in CON rats.  and , NS and WDR neurons, respectively, in CAP rats.

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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Fig. 2. Time courses of the mean spontaneous activity of caudalis NS neurons after bicuculline methiodide (BIC; 3 µg i.t.) application. Arrow represents the injection time (0 min).  and , mean ± SE of NS neurons in CON (n = 6) and CAP (n = 6) rats, respectively. *Significant difference between values pre- and post-BIC application (P < 0.05, repeated measures ANOVA followed by Student-Newman-Keuls method). #Significant difference in values at 5 min after BIC application between CAP and CON groups (P = 0.002, H = 35.68, Kruskal-Wallis ANOVA on ranks followed by Student-Newman-Keuls method). These symbols denoting statistical significance also apply to the following figures.


                              
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Table 1. Effects of intrathecal application of BIC or SAC on spontaneous activity and RF properties of caudalis nociceptive neurons in CAP rats and CON rats

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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Fig. 3. Time courses of the mean pinch mechanoreceptive field (RF) size of caudalis NS neurons after BIC (3 µg i.t.) application. Arrow represents the injection time (0 min).  and , means ± SE of NS neurons in CON (n = 6) and CAP (n = 6) rats, respectively. Significant differences in values were found between CON and CAP groups (P < 0.02, F1,40 = 6.329, 2-way ANOVA), and differences in values at corresponding 5- and 10-min time points between these 2 groups were also found significant (P < 0.05, Student-Newman-Keuls method). In addition, significant differences in values between baseline and 5-10 min after BIC application in both CON and CAP groups were found (P < 0.001 and P = 0.002, respectively, repeated measures ANOVA). Inset: examples of caudalis NS neuronal RF sizes delineated by pinch stimuli. In CON rat, region encompassing filled area indicates the RF before BIC application; filled plus shaded (touch-sensitive) areas indicate the RF after BIC application; in CAP rat, the pinch RF size (filled area) was reduced after BIC application.

The Vc NS neurons in CON rats did not show any noticeable responses to light tactile stimulation of their RF in resting conditions. However, they all showed tactile-evoked responses after BIC application; these responses appeared a few minutes after drug application and lasted for ~20 min (Fig. 4A). These novel tactile-sensitive areas (3.79 ± 1.11 cm2; P < 0.05) were located within the neuronal pinch RF and had a boundary that was not as sharp as that in WDR neurons, as reported previously (Chiang et al. 1998). Similarly, in all six NS neurons in CAP rats, a novel tactile RF area (2.61 ± 1.26 cm2; P < 0.05) appeared after BIC application and lasted for 30 min (Fig. 4A, Table 1). All five WDR neurons in both CAP and CON rats showed enlargement of the tactile RF to 605 ± 209% (n = 3) and 508 ± 143% (n = 2), respectively, of the control values, after BIC application. No significant difference between CAP and CON rats was found in tactile RF size of either WDR or NS neurons.



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Fig. 4. A: time courses of the reversible appearance of tactile RF in caudalis NS neurons after BIC (3 µg i.t.) application. Arrow represents the injection time (0 min).  and , means ± SE of NS neurons in CON (n = 6) and CAP (n = 6) rats, respectively. Significant differences were found in values between baseline and 5 min after BIC in CON rats and between baseline and 5-20 min in CAP rats (P < 0.001; F3,40 = 6.995, 2-way ANOVA followed by Student-Newman-Keuls method, P < 0.05), but not between CON and CAP groups (P > 0.6). B: time courses of the mean mechanical threshold of caudalis NS neurons after BIC (3 µg i.t.) application. Arrow represents the injection time (0 min).  and , means ± SE of NS neurons in CON (n = 6) and CAP (n = 6) rats, respectively. Significant differences were found in values between CON and CAP groups (P < 0.001, H = 58.24, Kruskal-Wallis ANOVA on ranks), and differences in values at all corresponding time points between these 2 groups were also significant by post hoc multiple comparison (#P < 0.05, Student-Newman-Keuls method). In addition, differences in values between baseline and 5-20 min in CON rats and between baseline and 5-30 min in CAP rats were also significant (P < 0.05, repeated measures ANOVA followed by Student-Newman-Keuls method).

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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Fig. 5. Time courses of the mean pinch response of caudalis NS neurons after BIC (3 µg i.t.) application. Arrow represents the injection time (0 min).  and , means ± SE of NS neurons in CON (n = 6) and CAP (n = 6) rats, respectively. Note that significant differences in pinch responses between baseline and 5-min time points were found in CON rats, but not in CAP rats (P < 0.05, F7,80 = 2.324, 2-way ANOVA). Differences in values between CON and CAP groups were also significant (P < 0.01, time × group, F7,80 = 2.923, 2-way ANOVA followed by Student-Newman-Keuls method).

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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Fig. 6. Time courses of mean spontaneous activity (A), tactile RF size (B), pinch RF size (C), mechanical threshold (D), and responses to pinch stimuli (E) of caudalis nociceptive neurons after 2-hydroxysaclofen (SAC; 4 µg i.t.) application. Arrow represents the injection time (0 min).  and , means ± SE of nociceptive (3 NS, 3 WDR) neurons in CON and those of other 3 NS and 3 WDR neurons in CAP rats, respectively. No significant differences were found in values between the 2 groups or between the values at baseline and at any time point after SAC application in either CON and CAP group.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha -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.


    ACKNOWLEDGMENTS

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.


    FOOTNOTES

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.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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