Introduction

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Convergent lines of evidence suggest that glycine and -aminobutyric acid (GABA) play important roles in the spinal processing of low-threshold mechanoreceptive input (Curtis et al., 1968; Yaksh, 1989; Todd, 1990; Todd and Sullivan, 1990; Sherman and Loomis, 1994; Sorkin et al., 1998). Glycinergic neurons in laminae II and III receive a major monosynaptic input from myelinated primary afferent fibers in the rat (Todd, 1990). GABA and glycine are colocalized in interneurons in spinal laminae I-III (Todd and Sullivan, 1990; Mitchell et al., 1993; Todd et al., 1996) and are coreleased from the same interneurons (Jonas et al., 1998). Furthermore, spinal glycine- and GABA_A receptors are localized on the same postsynaptic membranes (Mitchell et al., 1993; Todd et al., 1996), suggesting that GABA and glycine work together to effect sensory modulation.

Indeed, the blockade of spinal glycine- or GABA_A receptors yields an abnormal state suggestive of allodynia (a condition in which normally innocuous stimuli produce severe pain). Thus, intrathecal (i.t.) bicuculline (BIC; GABA_A antagonist) or strychnine (STR; glycine antagonist) produces a reversible state in which light touch evokes nociceptive-like vocalization, biting, and escape behavior in conscious rats and mice, and tactile-evoked autonomic, motor, and neurochemical responses in anesthetized rats (Yaksh, 1989; Minami et al., 1994; Sherman and Loomis, 1994; Milne et al., 1996; Onaka et al., 1996). Moreover, genetic variants such as the Poll Hereford calf (Gundlach et al., 1988) and the spastic mouse (White and Heller, 1982), which exhibit up to a 10-fold decrease in STR binding in the spinal cord, display exaggerated sensitivity to even modest cutaneous stimulation. These animal data are consistent with reports of pronounced hypersensitivity and pain to light touch in humans during STR intoxication (Arena, 1979), suggesting that glycine, and presumably GABA, are essential in the modulation of low-threshold mechanical input.

These observations are supported by previous electrophysiological data. Iontophoretic delivery of glycine and GABA diminished 1) the responsiveness of dorsal horn neurons to light tactile stimulation in cats; 2) the size of their cutaneous receptive fields (Zieglgänsberger and Hertz, 1971); and 3) the glutamate- and pinch-evoked firing of the spinothalamic tract neurons in monkeys (Wilcockson et al., 1984). Conversely, STR or BIC in the lumbar spinal cord diminished the inhibition elicited by natural or electrical stimulation of low-threshold afferent fibers of the cat (Game and Lodge, 1975), and increased neuronal activity evoked by low-threshold afferent input in cats and monkeys (Khayyat et al., 1975; Yokota et al., 1979). The effect of BIC on the electrophysiological activity of rat spinal neurons is dose-dependent (Sorkin et al., 1998). The blockade of inhibitory postsynaptic potentials (IPSPs) with BIC or STR or both enhanced the excitatory postsynaptic potentials of rat ventrolateral medullary neurons (Lin et al., 1998). To the extent that spinal GABA and glycine are necessary to balance the degree of excitation of convergent neurons, the removal of these inhibitory systems would strengthen the synaptic connections between non-nociceptive fibers and pain-signaling pathways. This could result in the miscoding of an innocuous stimulus as pain.

At the cellular level, there are important differences between GABA- and glycine-mediated inhibition. For example, the time course of IPSPs elicited by GABA and glycine are distinct. Short IPSPs (mean latency 3.6 ms, half-decay 11 ms) are mediated by glycine, whereas the longer IPSPs (mean latency 3.7 ms, half-decay 42 ms) are GABA mediated (Yoshimura and Nishi, 1995). Thus, glycine and GABA appear to have distinct but complementary effects on spinal neurons. There are also important differences in the spinal pharmacology of allodynia induced by glycine- and GABA antagonists. STR-allodynia in mice was blocked by the 1) nitric-oxide synthase inhibitor N-nitro-L-arginine methyl ester; 2) N-methyl-D-aspartate receptor antagonists AP-5 and ketamine; 3) non-N-methyl-D-aspartate receptor antagonist 6-cyano-2,3-dihydroxy-7-nitroquinoxaline; and 4) guanylate cyclase inhibitor methylene blue (Onaka et al., 1996). STR-allodynia was unaffected by the metabotropic receptor antagonists L-AP3 and L-AP4 (Onaka et al., 1996). In contrast, BIC-allodynia was blocked by L-AP3, L-AP4, and methylene blue but unaffected by N-nitro-L-arginine methyl ester, 6-cyano-2,3-dihydroxy-7-nitroquinoxaline, AP-5, and ketamine (Onaka et al., 1996).

If GABA acting at spinal GABA_A receptors, and glycine acting at STR-sensitive glycine receptors, negatively modulate low-threshold transmission through distinct but complimentary mechanisms, then the removal of both inhibitory systems should exaggerate an allodynic state. This outcome and the interaction between glycine and GABA in the spinal cord have not been determined. In the present study, we investigated the combined effects of i.t. STR and BIC using isobolographic analysis. We also verified the role of GABA_A receptors in this model.

	Materials and Methods

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Animals. Male, Sprague-Dawley rats (300-490 g at the time of the experiments) were obtained from the Vivarium, Animal Care Services, Memorial University of Newfoundland (St. John's, Canada). Animals were housed in the Animal Care Facility, with a 12-h dark/light cycle, a room temperature of 22°C, and free access to food and water. All experiments were conducted in accordance with the guidelines of the Canadian Council on Animal Care and were approved by the Memorial University Animal Care Committee.

Procedures. Under halothane anesthesia, rats were fitted with i.t. catheters prepared from stretched polyethylene tubing (PE-10 pulled to 1.5× the original length). The catheters, filled with sterile saline (Astra Pharma Inc., Mississauga, Canada), were inserted through the cisterna magna into the spinal subarachnoid space, and guided 8.5 cm caudally terminating near the L1-L2 segment. A fixed loop near the rostral end of the catheter was sutured to the overlying muscle to secure the catheter and the incision closed. The rostral tip was externalized on the top of the head and sealed with a stainless steel plug. Animals recovered for at least 4 days before experimentation and only those exhibiting normal motor, grooming, and feeding behavior were used.

Drugs. Bicuculline methiodide and strychnine hemisulfate were obtained from Sigma Chemical Co. (St. Louis, MO). Muscimol hydrobromide and R-(+)-baclofen hydrochloride were obtained from Research Biochemicals International (Natick, MA). Muscarine chloride, picrotoxin, and scopolamine hydrobromide were obtained from Aldrich Chemical Co. (Milwaukee, WI). All drugs were dissolved in 0.9% saline (Astra Pharma Inc., Mississauga, Canada) and administered i.t. in a volume not exceeding 10 µl. Drug solutions were flushed through catheter with 10 µl of saline.

Experimental Protocols. Dose-response curves for BIC and STR + BIC were determined in separate groups of rats. Each animal received i.t. saline, followed 10 min later by i.t. BIC (0.1, 0.25, 0.5, and 0.75 µg) or BIC:STR (0.001:0.1, 0.005:0.5, 0.0075:0.75, 0.01:1, and 0.1:10 µg). A fixed molar ratio of 1:133 was selected from the estimated ED₅₀ values for BIC and STR. The STR dose-response curve (data not shown) was constructed as previously published (Sherman and Loomis, 1994). This curve has been generated several times in our laboratory and has proven highly reproducible. The HD stimulus was applied at 5-min intervals until evoked cardiovascular/motor responses were no longer observed (approximately 30-45 min after BIC/STR injection). The total period of time that motor responses could be evoked by HD was also determined. The percentage of synchrony was calculated by determining the number of 1-min intervals with a synchronous EEG (determined visually) and expressing this as percentage of the experimental time.

Effect of Intrathecal Muscimol and Baclofen on STR-Allodynia. To establish the control responses to STR + HD (e.g., in absence of any other drug treatment), each rat received i.t. vehicle followed 10 to 15 min later by i.t. STR (40 µg). This dose of STR elicits optimal allodynia without convulsive activity in lightly anesthetized rats (Sherman and Loomis, 1994). Approximately 1 h later, rats were pretreated with i.t. muscimol (0.1, 0.5, 1, and 2.5 µg) or baclofen (1, 5, and 10 µg) followed 15 min later by i.t. STR (40 µg). Hair deflection was then applied at 5-min intervals. All animals received multiple drug doses. The order of dosing was always from low to high to reduce the possibility of a carry-over effect and thus, the recovery time. No more than three doses were administered to the same animal. Preliminary time course studies indicated that a 15-min pretreatment time was optimal for i.t. drug administration and that 1.5 h between doses was sufficient for complete recovery from even the highest doses.

Data Analysis. All cardiovascular data are presented as the maximum change in MAP (=systolic blood pressure +  pulse pressure) and HR evoked by HD relative to the immediate (1-min) prestimulus control period. Dose-response data were analyzed using regression analysis and ED₅₀ and 95% confidence interval (CI) values determined. Variability associated with single measurements is indicated by the S.E.M., whereas variability associated with blocks of data is indicated by the pooled 95% CI. Significant differences among the baclofen-treated groups were determined using completely randomized one-way ANOVA and the Newman-Keuls test. Methods of data analysis were based on a general text (Tallarida and Murray, 1987).

	Results

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Stroking the hair on the legs, flanks, and lower back of the lightly anesthetized animal with a cotton tip applicator was without effect in i.t. saline-treated animals. An identical stimulus applied to rats pretreated with i.t. STR and/or BIC evoked a progressive rise in HR and BP that persisted beyond the duration of the HD stimulus. These responses were accompanied by abrupt motor responses (scratching activity at the site of stimulation, kicking of the hind limbs, and muscular contraction in the affected dermatomes) and desynchrony of the EEG. Normalization of the EEG (return to a synchronous pattern), which occurred 1 to 2 min after the discontinuation of the HD stimulus, coincided with recovery of baseline BP and HR. The magnitude of these HD-evoked cardiovascular responses were dependent on the dose of BIC or the BIC + STR combination (Figs. 1 and 2). The cardiovascular and motor responses were only evoked by HD at discrete sites corresponding to dermatomes innervated by spinal segments near the site of STR and/or BIC injection. BIC-allodynia lasted up to 45 min following the injection of 0.75 µg compared with STR-allodynia, which persisted up to 30 min at a dose of 40 µg. At low doses, the STR:BIC combination exhibited a time course profile similar to that of STR-allodynia. At high doses, the combination resembled that of BIC-allodynia.

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Intrathecal bicuculline induces dose-dependent allodynia in lightly anesthetized rats. Dose-response curves illustrating the relationship between intrathecal bicuculline (alone) and the hair deflection-evoked heart rate (A) and mean arterial blood pressure responses (B). Each point represents the mean ± S.E.M. of six to eight rats.

View larger version (21K): [in this window] [in a new window]   Fig. 2.   Intrathecal bicuculline and strychnine combination induces dose-dependent allodynia in lightly anesthetized rats. Representative dose-response curves illustrating the relationship between intrathecal BIC and STR combination and the hair deflection-evoked heart rate (A) and mean arterial pressure responses (B). Note that the x-axis is labeled with only STR doses. However, each STR dose was combined with BIC in the ratio of 1:100 (molar ratio of 1:133) and the combined doses were administered. Each point represents the mean ± S.E.M. of six to eight rats.

Unlike i.t. STR, i.t. BIC also elicited spontaneous (nonevoked) increases in BP and HR, and motor responses (kicking and scratching of the affected area) that lasted up to 15 min after injection. These were especially noticeable at the highest dose (0.75 µg). In cases where spontaneous responses lasted longer than 5 min, the HD stimulus was withheld until the spontaneous rise in BP and HR began to normalize.

STR and BIC exhibited a significant difference in potency in this preparation. As shown in Table 1, the ED₅₀ values for i.t. BIC were 0.5 µg for MAP and 0.6 µg for HR. In contrast, the potency of i.t. STR was 25 µg for MAP and 37 µg for HR. The ED₅₀ values for BIC:STR combination were 26 ng:2.6 µg for MAP and 34 ng:3.4 µg for HR (Table 1). Isobolographic analysis revealed a significant supra-additive interaction between i.t. BIC and STR in this model (Fig. 3).

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Intrathecal strychnine and bicuculline combination results in supra-additive induction of allodynia. Isobolograms illustrating the supra-additive interaction between intrathecal BIC and STR in the induction of allodynia, based on the hair deflection-evoked HR (A) and MAP (B) responses in the rat. The two points each on x- and y-axes are the corresponding ED50 values obtained from the dose-response curves of i.t. STR alone and BIC alone. The dotted line is the line joining the corresponding confidence interval of these ED50 values. The third point plotted is the ED50 value with the corresponding confidence interval for STR and BIC combination. The fact that this point lies well below the CI of the individual ED50 values suggests a synergistic effect of the combined drug administration. [ED50 values for STR were determined previously (Sherman and Loomis, 1994).]

The allodynic effect of BIC was not mimicked by i.t. muscarine (2-4 µg, n = 3) nor was it affected by pretreatment with i.t. scopolamine (acetylcholinesterase inhibitor, 30 µg, n = 3) (data not shown). In the presence of i.t. picrotoxin (2 µg, n = 3), HD evoked cardiovascular and motor responses comparable to those of BIC (0.25 µg) (Table 2). Weak responses were also observed in absence of tactile stimulation (data not shown).

Intrathecal muscimol (0.1, 0.5, 1, and 2.5 µg), given 15 min before STR (40 µg), dose dependently inhibited all indices of STR-allodynia (Fig. 4). The ED₅₀ values and 95% CI for i.t. muscimol were 0.5 µg (0.4-0.7) for MAP, 0.5 µg (0.3-0.7) for HR, and 0.4 (0.3-0.6) for duration of motor responses. This antiallodynic effect was accomplished without a change in the percentage of synchrony of the EEG, which remained below the 60% cut-off. Baseline cardiovascular responses were also unaffected by muscimol up to 2.5 µg i.t. (data not shown). In contrast, baclofen (up to 10 µg i.t.) failed to inhibit STR-allodynia (Table 4). However, motor responses to HD were significantly attenuated by i.t. baclofen (*p < 0.01). Indeed, one rat receiving the 1-µg dose, and all rats receiving 5 µg exhibited reversible hind limb paralysis as determined by the absence of the pinch-evoked motor reflex.

View larger version (37K): [in this window] [in a new window]   Fig. 4.   Intrathecal muscimol dose dependently suppresses intrathecal STR-allodynia. Following a 15-min pretreatment with i.t. muscimol, responses to HD were determined in the presence of i.t. STR (40 µg). The HD-evoked increase in MAP (A), heart rate (B), the duration for which withdrawal response could be evoked (C), and the percentage of synchrony in the EEG (D) are shown. Each point represents mean ± S.E.M. of five to seven animals. Least-squares regression lines and the corresponding 95% CI (dotted lines) are shown. The horizontal solid lines and adjacent dotted lines indicate the mean ± 95% CI of all STR treatments (n = 23-24) before muscimol injection.

	Discussion

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Strychnine- or Bicuculline-Induced Allodynia. Hair deflection, applied to the affected dermatomes of rats pretreated with i.t. STR and/or BIC, evoked a marked cardiovascular and motor response, and desynchrony of EEG suggestive of allodynia. These data are consistent with previous studies (Yaksh, 1989; Sherman and Loomis, 1994) and indicate that robust allodynia can be selectively induced with i.t. STR and/or BIC in animals whose somatosensory systems are otherwise normal. Interestingly, the ED₅₀ of i.t. BIC was approximately 70 times lower than that of i.t. STR. There are a number of possibilities to explain this difference. The first is the more widespread distribution of GABA-containing neurons throughout the dorsal gray compared with glycine-containing elements (Todd and Sullivan, 1990). For example, of the total neurons counted in lamina I-III, approximately 35% were GABA immunoreactive, whereas only 17% were glycine immunoreactive (Todd and Sullivan, 1990; Laing et al., 1994). First, considering the fact that virtually all glycine immunoreactivity in lamina I-III is restricted to neurons that also contain GABA, these results suggest that only about 50% of total GABA-immunoreactive neurons cocontain glycine (Todd and Sullivan, 1990). Second, BIC-allodynia is longer lasting (up to 40 min) compared with STR-allodynia (30 min). This suggests a difference in the kinetics (clearance) of STR and BIC from the cerebrospinal fluid. In fact, the allodynic effects of STR disappeared rapidly (1-2 min) after discontinuation of the spinal infusion of STR (5-8 µg/min) in the rat (Sherman, 1994). However, a difference in clearance only partially explains the difference in the potency of BIC since this difference persists when STR or BIC is applied topically to the surface of the spinal cord (Zhang et al., 2001). The difference in potency may also reflect differences in the mechanism of receptor blockade. Unlike STR, BIC is a noncompetitive antagonist and thus does not compete with endogenous GABA (Takeuchi and Onodera, 1972; Shank et al., 1974). BIC is also metabolized to a pharmacologically active metabolite, bicucine (Olsen et al., 1976); the metabolites of STR are inactive (Oguri et al., 1989). This may also account for the longer duration of action of BIC compared with STR. Finally, GABA_A receptors, unlike glycine receptors, are present on both pre- and postsynaptic membranes in the dorsal horn (Bohlhalter et al., 1996). The concurrent blockade of pre- and postsynaptic receptors by BIC would not only be expected to cause increased excitation of second order neurons in response to low-threshold light tactile input but also to disinhibit the primary afferent neurons (Gmelin and Zimmermann, 1983; Desarmenien et al., 1984). This would facilitate spontaneous neuronal activity, thereby increasing the tonic release of excitatory amino acids in to the synaptic pool. Intrathecal BIC, but not STR, has been shown to induce the transient release of glutamate from the spinal cord of the rat (54% increase above baseline in the first 10 min after injection) (Ishikawa and Yaksh, 1996). This spontaneous activity could also explain the exaggerated responses observed in the absence of tactile stimulation.

Selective Role of GABA_A Receptors in the Induction of Allodynia. The i.t. administration of cholinergic agonists has been shown to induce spontaneous responses (i.e., increased pressor responses, tremor, scratching, tail biting, and chewing responses in conscious rats) similar to those evoked by HD in experimental allodynia (Magri and Buccafusco, 1988). In this regard, BIC is known to possess weak, reversible anticholinesterase activity, although this effect is normally observed in vitro at the concentrations of 30 µM (Svenneby and Roberts, 1973). GABA_A receptor blockade is observed at concentrations of <3 µM (Olsen et al., 1976). To exclude the possible role of acetylcholinesterase inhibition in BIC-allodynia, we examined the effect of i.t. 1) muscarine, 2) scopolamine, and 3) picrotoxin. Given that scopolamine failed to block the allodynic effect of BIC, and that i.t. muscarine had no allodynic activity, BIC-allodynia appears to be due to the blockade of GABA_A receptors. This conclusion is further supported by the allodynia-like behavior induced by i.t. picrotoxin, which blocks the chloride channel comprising the GABA_A receptor-channel complex. Picrotoxin lacks anticholinesterase activity (Svenneby and Roberts, 1973).

Supra-Additive Interaction between STR and BIC in Allodynia. From the discussion described above it is clear that i.t. BIC and STR each elicit acute and selective allodynia in the rat. This observation and qualitative similarity of their effects indicate the importance of glycine and GABA in the modulation of low-threshold afferent input. In combination, the doses of BIC and STR required to induce allodynia were 10 times lower than their individually effective doses. Isobolographic analysis clearly indicated a supra-additive interaction between STR and BIC. Such an interaction is consistent with the fact that 1) GABA_A and glycine receptors effect the opening of distinct chloride channels to hyperpolarize neurons (Bormann et al., 1987); 2) GABA_A and glycine receptors are co-contained on the same postsynaptic membranes in the spinal dorsal horn (Todd et al., 1996); 3) glycine and GABA appear to be coreleased from the same interneurons (glycine immunoreactivity is virtually restricted to neurons that also exhibit GABA immunoreactivity in the laminae I-III of the dorsal horn) (Todd and Sullivan, 1990; Mitchell et al., 1993); 4) vesicular transporters at inhibitory synapses accept both glycine and GABA as substrates (Burger et al., 1991; Sagne et al., 1997); and 5) GABA_A- and glycine-mediated inhibition of single spinal cord neurons have unique time courses (Game and Lodge, 1975; Baba et al., 1994; Yoshimura and Nishi, 1995).