Inhibitory effects of spinal baclofen on spinal dorsal horn neurones in inflamed and neuropathic rats in vivo

Brain Research 987 (2003) 67–75 www.elsevier.com / locate / brainres

Research report

Inhibitory effects of spinal baclofen on spinal dorsal horn neurones in inflamed and neuropathic rats in vivo David M. Sokal, Victoria Chapman* School of Biomedical Sciences, E Floor, University of Nottingham, Medical School, Nottingham NG7 2 UH, UK Accepted 3 July 2003

Abstract g-Aminobutyric acid (GABA) is a major inhibitory neurotransmitter, which modulates afferent transmission of nociceptive information at different levels of the central nervous system. Plasticity of spinal GABAergic systems may contribute to aberrant nociceptive responses associated with inflammatory and neuropathic pain states. Here potential changes in spinal GABA B receptor function in rats with peripheral inflammation and nerve injury, compared to control were investigated. Extracellular recordings of electrically evoked responses of spinal dorsal horn neurones were made in halothane anaesthetised rats. Effects of spinal administration of the GABA B receptor agonist baclofen (0.1–10 mg / 50 ml) on evoked responses of spinal neurones in control, hindpaw carrageenan inflamed, spinal nerve ligated and sham-operated rats were studied. In all groups of rats, spinal baclofen significantly reduced Ab-, Ad- and C-fibre evoked responses of spinal dorsal horn neurones in a dose related manner. Spinal pre-administration of the GABA B receptor antagonist, CGP-35348 (30 mg / 50 ml) significantly blocked the inhibitory effects of baclofen on evoked neuronal responses in control rats. Estimated ED 50 values for each fibre type within experimental groups were calculated, a significant (P,0.05) difference between the values for Ab-fibre-evoked and C-fibre mediated post-discharge responses of spinal dorsal horn neurones in spinal nerve ligated rats is reported. This finding may reflect decreased sensitivity of Ab-fibre-evoked responses to baclofen, as well as an increased sensitivity of post-discharge responses to baclofen in spinal nerve ligated rats. Overall, we report that GABA B -receptor control of A- and C-fibre evoked responses of spinal neurones is not profoundly altered in models of inflammatory and neuropathic pain.  2003 Elsevier B.V. All rights reserved. Theme: Sensory systems Topic: Pain modulation, pharmacology Keywords: Baclofen; GABA B ; Inflammation; Neuropathic; Carrageenan; Spinal nerve ligation; Nociception

1. Introduction g-Aminobutyric acid (GABA) is a major inhibitory neurotransmitter in the central nervous system and modulates afferent transmission of nociceptive information at different levels of the central nervous system, including the spinal cord (see Ref. [12]). Inhibitory effects of GABA are mediated mainly by two distinct classes of receptor, GABAA and GABA B . GABA B -receptors are heterodimers with subunits designated GABA B1 and GABA B2 [4]. Spinal GABA B -receptors are located at pre- and postsynaptic sites to primary afferent fibre endings. GABA B *Corresponding author. Tel.: 144-115-9709-459; fax: 144-115-9709259. E-mail address: [email protected] (V. Chapman). 0006-8993 / 03 / $ – see front matter  2003 Elsevier B.V. All rights reserved. doi:10.1016 / S0006-8993(03)03255-4

receptors are present on glutamatergic nerve endings of the spinal cord (see Ref. [22]) and capsaicin-sensitive primary afferent terminals [28,37]. A major function of GABA B receptors is to inhibit neurotransmitter release, in particular GABA, glutamate and substance P [19,34]. Post-synaptic GABA B -receptors [7] hyperpolarise membranes of dorsal horn neurones [18,37]. Blockade of spinal GABA B -receptors with the selective antagonist CGP-35348 facilitates C-fibre evoked responses of spinal neurones in control rats [31] and induces mechanical touch evoked pain (allodynia) in control rats [15], indicating the importance of tonic GABA B -receptor control of spinal nociceptive processing. Following sustained peripheral nociceptive input (formalin injection) there is an increase in spinal GABA B R1 and R2 gene expression [24]. By contrast, neurectomy of the sciatic nerve and Complete Freund’s adjuvant (CFA)-

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induced inflammation are associated with decreased GABA B -receptor binding in lamina II of the spinal cord 2–4 weeks following injury [5]. Following partial nerve injury, a loss of spinal GABA immunoreactivity [17] and reduced presynaptic GABA release [25] has been reported. Furthermore, partial nerve injury decreases dorsal horn levels of the GABA synthesising enzyme glutamic acid decarboxylase and induces neuronal apoptosis, resulting in a functional loss of GABAergic transmission in the superficial dorsal horn [25]. Density of GABA B -receptor binding sites and receptor affinity are, however, unaltered following partial nerve injury [30]. These data suggest that activation of spinal GABA B -receptors with exogenous agonists should be able to inhibit spinal nociceptive responses in models of partial nerve injury. Baclofen is a selective GABA B -receptor agonist [3], which is clinically used to alleviate spasticity associated with a range of conditions, including brain and spinal cord injury, multiple sclerosis and cerebral palsy (for review see [4,8]). The antinociceptive activity of baclofen in models of acute and chronic pain is well established [9,14,19,21,35]. Spinal baclofen inhibits mechanical hyperalgesia [27,30] and tactile allodynia [16,21] in nerve injured rats. Furthermore, Smith et al. [30] reported a 3-fold increase in sensitivity to systemic baclofen in neuropathic rats compared to control rats, although spinal receptor binding sites were unaltered (see above). Collectively, behavioural studies have provided strong evidence that activation of spinal GABA B -receptors reduces mechanical and thermal hyperalgesia associated with models of chronic pain states. The antinociceptive effects of baclofen are, however, complicated by motor dysfunction in the majority of behavioural studies. Electrophysiological studies of evoked responses of spinal neurones are not hampered by potential motor effects of drugs, and can identify potential changes of spinal GABA B -receptor function in relation to identified A- and C-fibre evoked responses. The aim of the present study was to use in vivo electrophysiological techniques to compare the effects of spinal baclofen on A- and C-fibre evoked responses of spinal neurones in a model of peripheral inflammation (hindpaw carrageenan injection) and nerve injury (spinal nerve ligation). Some of this work has been previously published as an abstract [32].

before electrophysiological recordings were made. A further control group received no intervention before electrophysiology (n56) and another group received a peripheral injection of carrageenan (n56) during the electrophysiological study.

2.1. Spinal nerve ligation model of neuropathy SNL rats were produced as previously described by Kim and Chung [20]. Male Sprague–Dawley rats (120–150 g) were anaesthetised using halothane (3% induction, 1.5% maintenance in 33% O 2 / 66% N 2 O; Fluothane, Zeneca, UK). A dorsal midline incision was made from approximately L3 to S2. The left paraspinal muscles were separated from the spinous processes at the L4-S2 level, the L4-L6 spinal nerves were identified, and part of the L6 transverse process was then removed. The L5 and L6 spinal nerves were isolated and tightly ligated with 6-0 silk thread just distal to the dorsal root ganglion and proximal to the formation of the sciatic nerve. Sham-operated rats underwent the same surgical procedure without ligation of the spinal nerves. Following surgery the rats were allowed to recover.

2.2. Behavioural testing After surgery, sham and SNL rats were group-housed, and post-operative (PO) posture and behaviour was closely monitored. Following SNL, the rats maintained good health and gained weight at a similar rate to sham-operated rats. From post-operative day 2 onwards, the rats were assessed behaviourally to monitor the development of mechanical allodynia every other day for 14 days. Testing was always performed between 08:30 and 11:00 h. Rats were placed in individual transparent Perspex cubicles with a wire mesh floor and were allowed a period of acclimatisation before testing began. Mechanical sensitivity of the ipsilateral and contralateral hindpaws was assessed by measuring the frequency of foot withdrawal to normally innocuous mechanical punctate stimuli. Stimuli were delivered, from below, to the plantar surface of the foot using 1, 4, and 10 g von Frey hairs. Each trial consisted of the application of a single von Frey hair repeated 10 times, tests were separated by 5 min.

2.3. Electrophysiology 2. Methods Electrophysiological experiments were performed on male Sprague–Dawley rats (240–300 g), which were group-housed in a light controlled room with free access to water and food. Rats were divided into one of four experimental groups. Two groups received either spinal nerve-ligation (SNL; n56) or sham-surgery (n56) and were allowed to recover for 2 weeks following surgery

Electrophysiological recordings were performed in control, carrageenan-inflamed, SNL and sham-operated rats. Methods are similar to those previously described by Chapman et al. [6]. Rats were anaesthetised with halothane (Fluothane, Zeneca, UK; 3% induction, 2% surgery, 1– 1.25% maintenance in 33% O 2 / 66% N 2 O) and a tracheal cannula was inserted. Rats were then placed in a sterotaxic frame to maintain stability during the recordings. A laminectomy was performed, lumbar vertebrae L1-L3 were

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located and partially removed and segments L4-L5 of the spinal cord were exposed. The spinal cord was held rigid by clamps rostral and caudal to the exposed section of spinal cord (L4 / 5) and a small well was formed with the surrounding muscle. Core body temperature was maintained at 36.5–37.5 8C by means of a heating blanket connected to a rectal temperature probe. Extracellular single-unit recordings of deep (500–1000 mm, laminae V–VI) convergent dorsal horn neurones were made with glass-coated tungsten microelectrodes [23]. The electrodes were descended vertically through the cord with a SCAT-01 microdrive (Digitimer, UK), depths of recorded neurones from the spinal cord surface were recorded. Single-unit activity was amplified and filtered (Digitimer, UK), signals were digitised and analysed using a CED micro1401 interface and Spike 2 data acquisition software (Cambridge Electronic Design, UK). Responses of wide dynamic range neurones following transcutaneous electrical stimulation of the receptive field were recorded. Neurones exhibited Ab-fibre and C-fibre evoked responses, which were elicited by a train of 16 stimuli (0.5 Hz, 2 ms pulse-width) set at three times the threshold for C-fibre evoked responses. Evoked responses were separated on the following criteria: Ab-fibre 0–20 ms; Ad-fibre 20–90 ms; and C-fibre 90–300 ms poststimulus. Responses evoked 300–800 ms post-stimulus were classified as the post-discharge of the neurone. Poststimulus histograms were constructed on-line. The nonpotentiated response was calculated as the number of C-fibre evoked and post-discharge action potentials produced by the first stimulation multiplied by 16. The nonpotentiated component of the C-fibre evoked response is reflective of the C-fibre input into the dorsal horn prior to facilitation of C-fibre evoked responses. Trains of electrical stimuli were repeated every 10 min, control responses (,10% variance) were recorded before spinal drug administration.

2.4. Carrageenan inflammation Following preparation for electrophysiological recording, l-carrageenan (100 ml 2% in saline; Sigma, UK) was injected into the plantar surface of the hindpaw of a single group of anaesthetised rats (n56). Electrical-evoked responses were recorded for 180 min at intervals of 10 min. The last three recordings of stable (,10%) evoked responses at ¯180 min post-carrageenan injection were taken as control values before spinal drug administration.

2.5. Drugs Drugs were applied directly onto the surface of the exposed segment (L4 / 5) of the spinal cord. Effects of spinal administration of baclofen (0.1–10 mg / 50 ml;8.9 mM–0.89 mM; dissolved in 0.9% saline; Tocris Cookson, UK) on electrically evoked responses of dorsal horn

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neurones in control, sham, spinal nerve ligated and carrageenan-inflamed rats (all n56) were assessed. Doses were selected on the basis of previous electrophysiological studies [9] and were given sequentially. Effects of each dose of baclofen were measured at 10-min intervals for 50 min post-drug administration. Preliminary studies indicated that inhibitory effects of the top dose of spinal baclofen lasted for up to 2.5 h (data not shown). For evaluation of receptor antagonism, the GABA B receptor antagonist CGP-35348 (30 mg / 50 ml;2.6 mM; Tocris Cookson, UK) was applied to the spinal cord 20 min before application of 3 mg / 50 ml baclofen in a further group of control rats (n54). The dose of CGP-35348 was selected on the basis of earlier behavioural [16] and electrophysiological [31] studies. In a separate group of anaesthetised rats (n54) the carotid artery was cannulated. Mean arterial blood pressure (MAP) was recorded prior to, and following spinal administration of baclofen (1, 3, 10 mg / 50 ml) at 10 min intervals for a duration of 50 min, for each dose of baclofen studied. Data are presented as the mean maximal effect6S.E.M. for each concentration. The minimum effective dose is defined as the minimum dose of baclofen that produces a significant effect compared to pre-drug control values. Statistical significance within groups compared to control was assessed using one-way ANOVA followed by a Dunnett’s post-hoc test, *P,0.05. Statistical significance between groups was assessed using one-way ANOVA followed by a Tukey’s post-hoc test.

3. Results

3.1. Development of mechanical allodynia in spinal nerve ligated rats Following tight ligation of L5 and L6 spinal nerves, spinal nerve ligated rats (n56) exhibited normal grooming behaviour and weight gain similar to the sham controls (n56). The development of mechanical allodynia was monitored in spinal nerve ligated rats over a 2-week period. Following application of non-noxious mechanical stimuli (von Frey filament, bending weight 10 g) to the ipsilateral paw, spinal nerve ligated rats exhibited a brisk withdrawal response. Mechanical allodynia was observed as early as PO day 2 and was maintained over the following 2-week period (data not shown). Sham-operated rats exhibited negligible levels of mechanical allodynia (data not shown).

3.2. Characteristics of neuronal populations of control, carrageenan-inflamed, spinal nerve ligated and shamoperated rats In vivo extracellular recordings of dorsal horn neurones

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Table 1 Mean depth, electrically evoked C-fibre latencies (CL ) and thresholds (CTH ) of dorsal horn neurones in control, sham operated, spinal nerve ligated (SNL) and carrageenan-inflamed rats in vivo

Depth (mm) CTH (mA) CL (ms)

Control

Inflamed

SNL

Sham

792674 1.7560.2 178619

877651 1.9560.2 192626

807655 2.2260.3 15669

880686 2.4260.3 181619

were carried out between post-operative days 13–17 in spinal nerve ligated and sham-operated rats. In vivo extracellular recordings were also made in control rats and in rats with an established hindpaw carrageenan-induced inflammation. The mean depths of neurones, thresholds

and latencies of C-fibre evoked responses were similar for the population of neurones recorded from control (n56), sham-operated (n56), spinal nerve-ligated (n56) and carrageenan-inflamed (n56) rats (Table 1). Control electrically evoked responses of dorsal horn neurones did not differ between the four groups of rats studied (Table 2). An example response of a single dorsal horn neurone following transcutaneous electrical stimulation of the peripheral receptive field is shown in Fig. 1.

3.3. Effects of baclofen in control rats In control rats, the minimum effective dose of spinal baclofen for Ab- and Ad-fibre evoked responses of spinal

Table 2 Control electrical-evoked firing of dorsal horn neurones in control, sham, spinal nerve-ligated (SNL) and carrageenan-inflamed rats

Control Inflamed SNL Sham

Ab-fibre

Ad-fibre

C-fibre

PD

NP

97618 96624 125623 128614

82623 77615 82630 56621

389698 441670 326661 363648

213647 339681 283676 235642

439691 356662 327667 244683

Data are the mean6S.E.M. number of action potentials evoked by a train of 16 electrical stimuli. PD, post discharge response; NP, non-potentiated C-fibre evoked response.

Fig. 1. (A) Example extracellular recording showing the response of a single dorsal horn to a train of transcutaneous electrical stimuli delivered to the receptive field on the hindpaw (16 electrical stimuli at 3 times C-fibre threshold, 2 ms pulse width, 0.5 Hz). (B) Response of the neurone to the first electrical stimulus, demonstrating the latency of evoked responses: Ab-fibre 0–20 ms; Ad-fibre 20–90 ms; C-fibre 90–300 ms. The neurone exhibited hyperexcitability 300–800 ms post-stimulation.

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neurones was 3 mg / 50 ml (F55.06, P,0.05 and 5.43, P,0.05, respectively, Fig. 2A,B). By contrast, the minimum effective dose of spinal baclofen for C-fibre evoked responses and non-potentiated responses of spinal neurones was 1 mg / 50 ml (F59.15, P,0.001 and F59.32, P,0.05, respectively, Fig. 2C,E). The minimum effective dose of spinal baclofen for C-fibre mediated post-discharge responses of neurones was 3 mg / 50 ml (F56.30, P,0.001, Fig. 2D). In a separate group of control rats (n54) the effect of spinal administration of the top three doses of baclofen (1, 3 and 10 mg / 50 ml) on MAP was measured. Control MAP in halothane anaesthetised rats was 6262.4 mmHg. Spinal administration of baclofen did not significantly alter MAP at any time point studied. Changes in MAP at 30 min following 1, 3 and 10 mg / 50 ml baclofen were 2.81 mmHg, 21.39 mmHg and 20.95 mmHg, respectively.

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3.4. Effects of baclofen in sham-operated and spinal nerve ligated rats In sham-operated rats, the minimum effective dose of spinal baclofen for Ab-fibre evoked responses of spinal neurones was 10 mg / 50 ml (F54.03, P,0.05. Fig. 2A). By contrast the minimum effective dose of spinal baclofen for Ad-fibre and C-fibre evoked responses of spinal neurones was 1 mg / 50 ml (F55.38, P,0.05, F514.9, P,0.001, respectively; Fig. 2B,C). The minimum effective dose of spinal baclofen for C-fibre mediated post-discharge responses and non-potentiated C-fibre responses of spinal neurones was 1 mg / 50 ml (F59.96, P,0.001 and F56.09, P,0.05, respectively, Fig. 2D,E). In spinal nerve ligated rats, the minimum effective dose of spinal baclofen for Ab-, Ad- and C-fibre evoked neuronal responses was 3 mg / 50 ml (F57.60, F58.11 and

Fig. 2. Effects of spinal administration of baclofen on (A) Ab-, (B) Ad- and (C) C-fibre, (D) C-fibre-mediated post discharge and (E) C-fibre-mediated non-potentiated responses of spinal neurones in control, nerve injured (SNL) and sham-operated rats. Data are presented as mean6s.e.m.

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F512.72, respectively, P,0.001 for all, Fig. 2A–C). Similarly, the minimum effective dose of spinal baclofen for C-fibre mediated post-discharge responses of neurones was 3 mg / 50 ml (F55.55, P,0.001 for both; Fig. 2D). Finally, the minimum effective dose of spinal baclofen for non-potentiated C-fibre responses of spinal neurones was 0.1 mg / 50 ml (F510.65, P,0.05, Fig. 2E).

receptor antagonist, CGP-35348 (30 mg / 50 ml), significantly (P,0.05) decreased the magnitude of the inhibitory effects of baclofen (3 mg / 50 ml) on Ab- and C-fibre evoked neuronal responses (Fig. 4). CGP-35348 attenuated inhibitory effects of baclofen on Ad-fibre evoked neuronal responses, significance was not reached.

3.5. Effects of baclofen in carrageenan-inflamed rats

4. Discussion

In carrageenan-inflamed rats, the minimum effective dose of spinal baclofen for Ab-fibre evoked responses of spinal neurones was 10 mg / 50 ml (F53.34, P,0.05, Fig. 3A). The minimum effective dose of spinal baclofen for Ad- and C-fibre evoked responses was 1 mg / 50 ml (F5 4.48, P,0.05 and F55.78, P,0.001, respectively, Fig. 3A). The minimum effective doses of spinal baclofen for C-fibre mediated post-discharge and non-potentiated Cfibre responses of neurones were 3 mg / 50 ml (F57.22, P,0.001, Fig. 3B) and 1 mg / 50 ml (F510.13, P,0.001, Fig. 3B), respectively.

Effects of spinal baclofen on evoked responses of spinal neurones were studied in control, nerve injured, shamoperated rats and rats with hindpaw carrageenan-inflammation. Nerve injured, but not sham-operated, rats manifested the temporal development of mechanical allodynia over a 2-week period, as previously described [6]. Electrophysiological studies of A- and C-fibre evoked responses of wide dynamic range dorsal horn neurones, which were not classified on the basis of morphology, or projection, were made. C-fibre thresholds, latencies and evoked responses of wide dynamic range dorsal horn neurones were similar for the four groups of rats studied. This finding corroborates data obtained in previous electrophysiological studies of spinal dorsal horn neurones in rats [6,33]. Spinal administration of baclofen significantly inhibited electrical-evoked responses of dorsal horn neurones in control, sham-operated, nerve-injured and carrageenan-inflamed rats. Comparisons between the inhibitory effects of baclofen on Ab-, Ad- and C-fibre evoked responses demonstrated similar trends for the four groups of rats studied. Comparison of the minimum effective dose of spinal baclofen suggested that Ab-fibre evoked responses were less sensitive to spinal baclofen, compared to Ad- and C-fibre evoked responses, for all groups of rats. However, significant differences in the estimated ED 50 s for baclofen on Ab-fibre evoked responses and C-fibre mediated postdischarge responses of spinal neurones were only observed in SNL rats. These differences may reflect decreased sensitivity of Ab-fibre evoked responses to baclofen, as well as an increased sensitivity of post-discharge responses to baclofen in nerve-injured rats. The time course of peak inhibitory effects of spinal baclofen reported here, is

3.6. Comparison of effects of baclofen between groups Mean maximal inhibitory effects of baclofen on evoked responses were observed at 40–50 min post drug administration in all groups of rats. Comparison of the estimated logED 50 values for each fibre type revealed no significant differences between control, carrageenan-inflamed, shamoperated and spinal nerve ligated rats. Comparison of the estimated log ED 50 values for each fibre type within experimental groups revealed a significant difference (P, 0.05, Kruskal–Wallis test and Dunn’s post hoc test) between the estimated log ED 50 value6S.E.M. for Abfibre (0.7860.18) and C-fibre mediated post-discharge responses (20.660.23) of dorsal horn neurones in spinal nerve ligated rats.

3.7. CGP-35348 blocks the inhibitory effects of baclofen in control rats Pre-administration (20 min) of the selective GABA B -

Fig. 3. Effects of spinal administration of baclofen on (A) Ab-, Ad- and C-fibre-evoked responses, and (B) C-fibre-mediated non-potentiated (NP) and C-fibre-mediated post-discharge (PD) responses of spinal neurones in carrageenan-inflamed rats. Data are presented as mean6s.e.m.

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Fig. 4. Pre-administration of the selective GABA B -receptor antagonist, CGP-35348 (30 mg / 50 ml), decreased the inhibitory effects of baclofen (3 mg / 50 ml) on (A) Ab- (B) Ad- and (C) C-fibre evoked neuronal responses in control (n54) rats. Data are presented as mean6s.e.m. Statistical significance between groups are assessed with a Mann-Whitney test, *P,0.05.

similar to that previously reported in behavioural studies [16,27]. Inhibitory effects of baclofen on evoked responses in control rats were blocked by spinal administration of the GABA B -receptor antagonist CGP-35348 [26], corroborating a previous report that CGP-35348 produces a dosedependent antagonism of the antinociception effect of spinal baclofen [13]. Thus, the mechanism underlying the inhibitory effect of baclofen on electrical-evoked responses of spinal neurones appears to be via GABA B -receptor activation. Spinal baclofen did not alter MAP in a control group of rats and, therefore, inhibitory effects of baclofen

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on evoked responses of spinal neurones are not likely to be secondary to cardiovascular effects of baclofen. GABA B -receptors are expressed on large and small diameter fibres, corresponding to A- and C-fibres, and post-synaptic neurones in the dorsal horn of the spinal cord. GABA B -receptors are predominantly localised in laminae I and II of the spinal cord, which plays a pivotal role in the relay and modulation of somatosensory input into the central nervous system. Presynaptic GABA B receptors inhibit calcium channels and reduce transmitter release and postsynaptic receptors hyperpolarise membranes of dorsal horn neurones [37]. Inhibitory effects of baclofen on A- and C-fibre evoked responses of deep spinal dorsal horn neurones, which receive inputs from superficial laminae, may be mediated by activation of both pre- and post-synaptic GABA B receptors. Our data corroborate an anatomical study that demonstrated presynaptic GABA B receptors on Ab-, Ad- and C-fibres [37]. However, a previous electrophysiological study in control rats [9] reported that C-, but not Ab-, fibre-evoked responses of spinal neurones are inhibited by spinal baclofen. The basis for this discrepancy between the results of these two electrophysiological studies is unknown. In spinal cord slices, C-fibre afferent transmission was reported to be more sensitive than Ad-fibre afferent transmission to baclofen [1]. Our in vivo data, however, demonstrate similar effects of baclofen on Ad- and C-fibre evoked responses of deep dorsal horn neurones, over the dose range studied. Differences in the findings of these two studies may reflect the contribution of post-synaptic, as well as pre-synaptic, GABA B receptors to the subsequent inhibition of Ad- and C-fibre evoked responses of deep dorsal horn neurones. Models of neuropathy are associated with plasticity of spinal receptor systems. Indeed, there is evidence for loss of spinal GABAergic inhibitory control, but not GABA B receptor density, following nerve injury (see Section 1). In the present study, spinal baclofen had similar inhibitory effects on A- and C-fibre evoked responses of spinal neurones in nerve injured rats and sham-operated controls. This finding corroborates reports of antinociceptive effects of similar doses of spinal baclofen in behavioural studies of neuropathic rats [21]. Thus in both behavioural and electrophysiological studies, activation of GABA B receptors with baclofen reduced noiciceptive responses in neuropathic rats, this is consistent with the maintained level of spinal GABA B receptor density under these conditions. Lower doses of spinal baclofen (0.3 and 1 mg) have also been shown to produce antiallodynic effects in nerve injured rats [16,27] and inhibit formalin evoked nociceptive behaviour [29]. The basis for differences in the effective doses of spinal baclofen reported in the present study and previous studies remains unknown, and may be related to the model and modality of noxious stimulus applied. Previously, increased sensitivity to systemic baclofen in neuropathic rats has also been reported [30]. Our

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data suggest that this increased sensitivity to systemic baclofen in neuropathic rats [30] may be mediated by sites of action other than the spinal cord. In all four groups of rats, C-fibre mediated post-discharge responses of spinal neurones were most sensitive to spinal baclofen. This finding suggests that baclofen is very effective at reducing spinal excitability evoked by repetitive C-fibre stimulation, which is associated with hyperalgesia in models of inflammation and nerve injury [36]. Hyperexcitable post-discharge responses are mediated by glutamatergic and peptidergic transmission and activation of excitatory amino acid receptors, including the NMDA and metabotropic glutamate receptors [10,11,36,38]. In vitro spinal cord studies of Ca 21 -dependent depolarization-induced overflow of endogenous glutamic acid and GABA demonstrated that baclofen inhibited overflow of glutamate, but was virtually inactive against that of GABA [2]. These data suggest that there are pharmacological differences in GABA B autoreceptors on GABA-releasing terminals and GABA B -receptors on glutamatergic terminals in the spinal cord [2] (see Refs. in [4]). By contrast, inhibitory actions of baclofen on spontaneous transmission in rat spinal cord substantia gelatinosa neurones are similar at GABA B -receptors on GABAergic and glutamatergic nerve terminals [18]. The ability of spinal baclofen to inhibit evoked glutamatergic transmission, without modulating spinal GABA release, may contribute to the marked effect of baclofen on electrically evoked C-fibre mediated post-discharge responses of spinal neurones in inflamed, nerve-injured and control rats reported in this study.

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Acknowledgements We gratefully acknowledge Dr. Michael Randall for his help in performing mean arterial blood pressure measurements. This study was supported by the Wellcome Trust.

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