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ALIMENTARY |
1 Department of Anaesthesiology and Pain Medicine, The University of Texas M. D. Anderson Cancer Center, Houston, TX 77030, USA
2 Department of Anaesthesiology, The Third Hospital of Hebei Medical University, Shijiazhuang 050051, China
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Abstract |
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-S included in the internal solution) on lamina II neurons in rat spinal cord slices. The frequency of glutamatergic mEPSCs and the amplitude of monosynaptic EPSCs evoked from the dorsal root were significantly higher in diabetic than in control rats. On the other hand, the basal frequency and amplitude of GABAergic spontaneous IPSCs and mIPSCs and those of glycinergic spontaneous IPSCs and mIPSCs did not differ significantly between control and diabetic rats. The GABAB agonist baclofen produced a significantly greater reduction in dorsal root-evoked EPSCs and the frequency of mEPSCs in control than in diabetic rats. However, the inhibitory effect of baclofen on GABAergic and glycinergic spontaneous IPSCs and mIPSCs was not significantly different in the two groups. These findings suggest that increased glutamatergic input from primary afferents to dorsal horn neurons may contribute to synaptic plasticity and central sensitization in diabetic neuropathic pain. Furthermore, the function of presynaptic GABAB receptors at primary afferent terminals, but not that on GABAergic and glycinergic interneurons, in the spinal cord is reduced in diabetic neuropathy.
(Received 4 December 2006;
accepted after revision 10 January 2007;
first published online 11 January 2007)
Corresponding author H.-L. Pan: Department of Anaesthesiology and Pain Medicine, Unit 409, The University of Texas M. D. Anderson Cancer Center, 1400 Holcombe Blvd, Houston, TX 77030, USA. Email: huilinpan{at}mdanderson.org
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Introduction |
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The GABAB receptor is widely distributed in the central nervous system (Chu et al. 1990), including the spinal lamina I–III in both humans and animals (Price et al. 1984; Waldvogel et al. 1990). In addition to their function as autoreceptors for feedback regulation of synaptic GABA release, presynaptic GABAB receptors also serve as heteroreceptors that regulate synaptic glycine and glutamate release to spinal dorsal horn neurons (Iyadomi et al. 2000; Li et al. 2002; Wang et al. 2006). Intrathecal administration of the GABAB receptor agonist baclofen produces antinociception in acute (Dirig & Yaksh, 1995) and chronic (Malcangio & Bowery, 1993; Smith et al. 1994) pain models. The GABAB receptor is also involved in the analgesic effect of muscarinic agonists (Chen & Pan, 2003b). Clinically, intrathecal administration of baclofen has been used as an adjuvant analgesic to treat patients with neuropathic pain (Fromm, 1994; Slonimski et al. 2004). However, it has been reported that the antinociceptive effect of baclofen is reduced in rats with diabetic neuropathy (Malcangio & Tomlinson, 1998). Notably, previous studies of the GABAB receptor have largely focused on its level in the spinal cord following nerve ligation (Smith et al. 1994; Castro-Lopes et al. 1995; Yang et al. 2004; Engle et al. 2006). The functional changes of GABAB receptors in the control of synaptic input to dorsal horn neurons in diabetes remain unclear. Therefore, in this study, we determined potential changes in excitatory and inhibitory synaptic input to spinal dorsal horn neurons and the role of presynaptic GABAB receptors in the regulation of synaptic transmission in the spinal cord in a rat model of diabetic neuropathy.
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Methods |
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Male Sprague-Dawley rats (4 weeks old) initially weighing 150–170 g were used. The experiments were carried out according to the guidelines laid down by the Animal Care and Use Committee of the University of Texas M. D. Anderson Cancer Center. All efforts were made to minimize both the suffering and number of animals used. Diabetes was induced by a single intraperitoneal injection of streptozotocin (STZ, 50 mg kg–1, Sigma) freshly dissolved in 0.9% sterile saline (Chen & Pan, 2002, 2003a). Diabetes was confirmed in STZ-injected rats by measurement of plasma glucose concentration in blood samples from the tail vein. The glucose level was assayed enzymatically using the Sigma diagnostic glucose reagents, and the colourimetric absorbance readings were obtained at 450 nm using a microplate spectrophotometer (SPECTRAmax Plus, Molecular Devices, Sunnyvale, CA, USA). Age-matched vehicle (saline) injected rats were used as the controls.
After STZ injection, rats developed persistent mechanical allodynia and hyperalgesia within 3 weeks (Courteix et al. 1994; Chen & Pan, 2002). Following confirmation of allodynia, final electrophysiological experiments were performed on rats 3–4 weeks after STZ injection. Tactile allodynia was assessed using a series of calibrated von Frey filaments (Stoelting Co., Wood Dale, IL, USA). They were applied perpendicularly to the plantar surface of both hindpaws with sufficient force to bend the filament for 6 s. Brisk withdrawal or paw flinching was considered as a positive response. In the absence of a response, the filament of the next greater force was applied. Following a response, the filament of the next lower force was applied. The tactile stimulus producing a 50% likelihood of withdrawal response was calculated by using the ‘up–down’ method, as previously described (Chen & Pan, 2002; Khan et al. 2002).
Spinal cord slice preparations
Rats were anaesthetized with 2–3% isoflurane in O2 delivered through a nose cone, and the lumbar segment of the spinal cord was rapidly removed through a limited laminectomy (Zhang et al. 2005; Wang et al. 2006). The need for supplemental doses during the surgery was assessed by the withdrawal reflex to paw pinch and by spontaneous movement. The rats were then killed by inhalation of 5% isoflurane. The lumbar spinal cord segment was immediately placed in ice-cold sucrose artificial cerebrospinal fluid (aCSF) presaturated with 95% O2 and 5% CO2. The sucrose aCSF contained the following (mM): 234 sucrose, 3.6 KCl, 1.2 MgCl2, 2.5 CaCl2, 1.2 NaH2PO4, 12.0 glucose, and 25.0 NaHCO3. After removal of the dura, the spinal cord segment was then glued on the stage of a vibratome (Technical Products International, St Louis, MO, USA). Transverse spinal cord slices (350 µm) were cut in the ice-cold sucrose aCSF and pre-incubated in Krebs solution continuously gassed with 95% O2 and 5% CO2 at 34°C for at least 1 h before they were transferred to the recording chamber. The Krebs solution contained (mM): 117.0 NaCl, 3.6 KCl, 1.2 MgCl2, 2.5 CaCl2, 1.2 NaH2PO4, 11.0 glucose, and 25.0 NaHCO3. The slice was placed in a glass-bottomed chamber (bath volume, 1.8 ml; Warner Instruments, Hamden, CT, USA) and fixed with parallel nylon threads supported by a U-shaped stainless steel weight. The slice was continuously perfused with Krebs solution at 5.0 ml min–1 at 34°C maintained by an inline solution heater and a temperature controller (TC-324; Warner Instruments).
Electrophysiological recordings
Postsynaptic currents were recorded using the whole-cell voltage-clamp techniques (Li et al. 2002; Zhang et al. 2005). The neurons located in the lamina II of the spinal cord were identified under a fixed-stage microscope (BX50WI, Olympus, Tokyo, Japan) with differential interference contrast/infrared illumination. The lamina II has a distinct translucent appearance and can be easily distinguished under the microscope. The electrode for the whole-cell recordings was pulled from borosilicate glass capillaries with a puller (P-97, Sutter Instrument, Novato, CA, USA). The impedance of the pipette was 3–5 M
when filled with internal solution. The internal pipette solution for recording of inhibitory postsynaptic currents (IPSCs) contained (mM): 110 Cs2SO4, 5 KCl, 2.0 MgCl2, 0.5 CaCl2, 5.0 Hepes, 5.0 EGTA, 5.0 ATP-Mg, 0.5 Na-GTP, 1 guanosine 5'-O-(2-thiodiphosphate) (GDP--S), and 10 lidocaine N-ethyl bromide (QX314); the solution was adjusted to pH 7.2–7.4 with 1 M CsOH (290–320 mosmol l–1). To record excitatory postsynaptic currents (EPSCs), 130 mM potassium gluconate was used to replace 110 mM Cs2SO4 in the pipette solution. GDP--S, a general G-protein inhibitor, was added to the internal pipette solution to block the possible postsynaptic effect mediated by GABAB receptors through G proteins (Li et al. 2002; Zhang et al. 2005). QX314 was added to the internal solution to suppress the action potential generation from the recorded cell. Postsynaptic currents were recorded using an amplifier (MultiClamp 700A, Axon Instruments, Foster City, CA, USA) at a holding potential of 0 mV for IPSCs and –70 mV for EPSCs. Signals were filtered at 1–2 kHz, digitized at 10 kHz, and stored on a Pentium computer with pCLAMP 9.0 (Axon Instruments). The evoked EPSCs (eEPSCs) from lamina II neurons were induced by electrical stimulation through a bipolar tungsten electrode placed in the dorsal root entry zone or the attached dorsal root (Li et al. 2002; Pan et al. 2002). To record the miniature IPSCs (mIPSCs) or miniature EPSCs (mEPSCs), 1 µM tetrodotoxin (TTX) was added to the perfusion solution. Input resistance and series resistence were continuously monitored during the entire period of data collection, and recordings were abandoned if these parameters changed more than 15%.
STZ, baclofen, GDP--S, strychnine, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), and bicuculline were obtained from Sigma-Aldrich (St Louis, MO, USA). TTX and QX314 were obtained from Alomone Laboratories (Jerusalem, Israel), and CGP55845 was purchased from Tocris (Ellisville, MO, USA). Each dose of baclofen was bath applied for 2 min. All drugs were dissolved in Krebs solution before the experiments and perfused into the slice chamber at final concentrations.
Data analysis
Data are presented as the mean ± S.E.M. Analysis of the effect of baclofen on the amplitude of eEPSCs was performed using Clampfit (Axon Instruments). The amplitude and frequency of EPSCs and IPSCs were analysed off-line using a peak detection program (MiniAnalysis; Synaptosoft, Leonia, NJ, USA). Detection of events was accomplished by setting a threshold above the noise level. The EPSCs and IPSCs were detected by the fast rise time of the signal over an amplitude threshold (typically 6–10 pA) above the background noise. We manually excluded the event when the background noise was erroneously identified as the IPSCs or EPSCs by the program. The background noise level was typically constant throughout the recording of a single neuron. The cumulative probability of the amplitude and inter-event interval of mIPSCs and mEPSCs were compared using the Kolmogorov-Smirnov test, which estimates the probability of two cumulative distributions being similar. At least 100 EPSCs and IPSCs were used in each analysis. The effect of baclofen on the amplitude of eEPSCs and the frequency of spontaneous IPSCs, mIPSCs and mEPSCs was determined using either two-way ANOVA with Bonferroni's post hoc test or repeated measures ANOVA. The IC50 value of baclofen and their 95% confidence limits were determined by non-linear regression analysis of the dose–response curves. P < 0.05 was considered to be statistically significant.
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Results |
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) and series resistance (21.3 ± 1.6 versus 22.6 ± 2.1 M; unpaired t test) of recorded lamina II neurons between control (n
= 52 neurons) and diabetic (n
= 57 neurons) rats. The evoked EPSCs of dorsal horn neurons and the baclofen effect in control and diabetic rats
GABAB receptors are located on the central terminals of primary afferents and dorsal horn neurons in the spinal cord (Price et al. 1984, 1987). To determine the role of presynaptic GABAB receptors in the regulation of synaptic glutamate release from primary afferents to dorsal horn neurons, the effect of the GABAB agonist baclofen on monosynaptic eEPSCs was tested. The eEPSCs were recorded from lamina II neurons in the presence of 10 µM bicuculline and 0.5 µM strychnine. The eEPSC was considered to be monosynaptic if (1) the latency was constant following electrical stimulation (0.2 Hz), and (2) there was no conduction failure or increased latency for monosynaptic eEPSCs when stimulation frequency was increased to 20 Hz (Li et al. 2002). We used a fixed stimulation intensity (0.2 ms and 0.5 mA) to evoke monosynaptic eEPSCs in both diabetic and control rats. Notably, the peak amplitude of monosynaptic EPSCs evoked from the dorsal root before drug application was significantly higher in diabetic than in control rats (Fig. 1A and B; unpaired t test). Bath application of baclofen for 2 min dose-dependently (1–50 µM) reduced the peak amplitude of monosynaptic eEPSCs in 13 neurons from control and 11 neurons from diabetic rats (Fig. 1A and B; repeated measures ANOVA test). At all the concentrations (1–50 µM) tested, baclofen produced a significantly greater effect on the amplitude of monosynaptic eEPSCs in non-diabetic control than diabetic rats (Fig. 1A and C; two-way ANOVA test). In all neurons tested, the eEPSCs were completely blocked by 10 µM CNQX, a non-NMDA glutamate receptor antagonist.
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To ensure the specific effect of baclofen on GABAB receptors, the highly selective GABAB receptor antagonist CGP55845 (Li et al. 2002; Wang et al. 2006) was used. In this protocol, the stimulation intensity (0.3 ms, 0.5 mA and 0.2 Hz) was fixed in the recording of monosynaptic eEPSCs in both diabetic and control rats. Bath application of 1 µM CGP55845 alone for 4–5 min before perfusion of baclofen did not significantly change the peak amplitude of eEPSCs. In the presence of 1 µM CGP55845, 50 µM baclofen failed to alter the amplitude of eEPSCs in 11 neurons from control rats and 13 neurons from diabetic rats (Fig. 1E). Notably, a more complete washout of the baclofen effect was observed in this protocol. This is probably due to a single dose of baclofen used in this protocol.
Glutamatergic mEPSCs of lamina II neurons and the baclofen effect in control and diabetic rats
We then determined the presynaptic effect of baclofen on spontaneous quantal (action potential-independent) release of glutamate in the spinal cord of diabetic and control rats. Glutamatergic mEPSCs were recorded in the presence of 10 µM bicuculline, 0.5 µM strychnine and 1 µM TTX. The baseline frequency of mEPSCs was significantly higher in diabetic (6.91 ± 0.93 Hz, n = 9 neurons) than in control rats (4.45 ± 0.46 Hz, n = 10 neurons, Fig. 2; unpaired t test). Bath application of 1–50 µM baclofen dose dependently decreased the frequency, but not the amplitude, of mEPSCs in both groups (Fig. 2A–C; two-way ANOVA test). The cumulative probability analysis of mEPSCs revealed that the distribution pattern of the inter-event interval of mEPSCs was shifted toward the right in response to baclofen. However, the distribution pattern of the amplitude of mEPSCs was not significantly changed (Kolmogorov-Smirnov test). When the effect was normalized to the baseline, baclofen caused a significantly greater decrease in the frequency of mEPSCs in the control than in diabetic rats (Fig. 2C; two-way ANOVA test). In all neurons examined, the mEPSCs were completely blocked by 10 µM CNQX, a non-NMDA receptor antagonist (Fig. 2A).
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GABAB receptors are also located on GABAergic and glycinergic inhibitory interneurons in the spinal cord dorsal horn (Iyadomi et al. 2000; Wang et al. 2006). To examine the role of presynaptic GABAB receptors in the control of synaptic GABA release to dorsal horn neurons in diabetes, we tested the dose–response effect of baclofen on GABAergic spontaneous IPSCs and mIPSCs in spinal lamina II neurons. GABAergic spontaneous IPSCs were recorded in the presence of 10 µM CNQX and 0.5 µM strychnine. The baseline frequency and amplitude of spontaneous IPSCs were not significantly different between the 14 neurons recorded from control and 14 neurons from diabetic rats (Fig. 3). Baclofen at the concentration of 1–50 µM dose dependently decreased the frequency, but not the amplitude, of GABAergic spontaneous IPSCs (Fig. 3; unpaired t test) in all neurons tested from control and diabetic rats. The cumulative probability analysis of IPSCs revealed that the distribution pattern of the inter-event interval of IPSCs was shifted toward the right in response to baclofen. However, the distribution pattern of the amplitude of IPSCs was not significantly altered (Kolmogorov-Smirnov test). The inhibitory effect of baclofen on GABAergic spontaneous IPSCs was not significantly different in the control and diabetic groups (Fig. 3; two-way ANOVA test).
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We next determined the role of presynaptic GABAB receptors in the control of glycinergic input to spinal dorsal horn neurons in diabetes. Glycinergic spontaneous IPSCs were recorded from the lamina II neurons in control and diabetic rats. Glycinergic spontaneous IPSCs were recorded in the presence of 10 µM CNQX and 10 µM bicuculline from additional neurons from control (n = 11 neurons) and diabetic (n = 9 neurons) rats. The baseline frequency and amplitude of spontaneous IPSCs of dorsal horn neurons were not significantly different between control and diabetic rats (Fig. 5A and B). The cumulative probability analysis of IPSCs showed that the distribution pattern of the inter-event interval of IPSCs was shifted toward the right in response to baclofen. However, the distribution pattern of the amplitude of IPSCs was not significantly changed (Kolmogorov-Smirnov test). Also, bath application of 1–50 µM baclofen inhibited the frequency of glycinergic spontaneous IPSCs similarly in both groups in a dose-dependent manner (Fig. 5C, two-way ANOVA test). Glycinergic spontaneous IPSCs were completely eliminated by 0.5 µM strychnine (Fig. 5A).
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Discussion |
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Spinal lamina II contains both excitatory and inhibitory interneurons and is an important region for the modulation of nociceptive information from primary afferent nerves (Cervero & Iggo, 1980; Yoshimura & Jessell, 1989; Yoshimura & Nishi, 1995; Pan & Pan, 2004). Glutamate is an important excitatory neurotransmitter released from central terminals of primary afferents (Yoshimura & Nishi, 1993; Pan & Pan, 2004). It is possible that the glutamatergic input to these neurons is increased in diabetic rats. In support of this hypothesis, we found that the amplitude of monosynaptic eEPSCs was much larger in diabetic than control rats when the dorsal root was stimulated electrically with the same intensity. This change may reflect increased glutamate release from the primary afferents, increased excitability of primary afferents, and/or increased expression of postsynaptic AMPA/NMDA receptors (Tomiyama et al. 2005; Daulhac et al. 2006) in the spinal cord in diabetic neuropathy. Since the frequency but not the amplitude of mEPSCs was significantly higher in diabetic than in control rats, it is likely that the increased glutamatergic input to dorsal horn neurons is due to increased glutamate release probability or increased number of release sites at the presynaptic terminals. Thus, enhanced glutamate release from primary afferent terminals to dorsal horn neurons in diabetic neuropathy may contribute to the hyperexcitability of dorsal horn neurons and maintenance of diabetic neuropathic pain (Chen & Pan, 2002). These data are consistent with previous studies showing that NMDA and non-NMDA antagonists are effective in the treatment of diabetic neuropathic pain (Calcutt & Chaplan, 1997; Malcangio & Tomlinson, 1998). This electrophysiological study provides new functional evidence for increased glutamatergic input from primary afferents to dorsal horn neurons in diabetic neuropathic pain.
GABA and glycine are the two important inhibitory neurotransmitters in the spinal cord (Yoshimura & Nishi, 1993; Li et al. 2002; Pan & Pan, 2004). We observed that the baseline frequency of GABAergic and glycinergic spontaneous IPSCs was not significantly different in the diabetic and control groups. Also, we found the basal frequency and amplitude of GABAergic mIPSCs and those of glycinergic mIPSCs were similar in the control and diabetic groups. It seems that GABAergic and glycinergic interneurons and their terminals impinging upon dorsal horn neurons are not significantly altered in diabetic neuropathy. On the other hand, in a neuropathic pain model induced by nerve ligation, presynaptic GABA (but not glycine) release onto lamina II neurons appears attenuated (Moore et al. 2002). It should be noted that although loss of GABAergic neurons has been suggested in the spinal cord after nerve ligation (Moore et al. 2002), this finding has been disputed by a more recent study showing that the density of neurons in laminas I–III is not reduced on the ipsilateral side after the nerve injury and that apoptotic cells in the spinal cord seem to be microglia (Polgar et al. 2005). Furthermore, it is possible that nerve ligation injury and diabetic neuropathy (due to metabolic disorders) represent distinct aetiologies that may affect differently GABAergic interneurons in the spinal cord. Consistent with the results in our previous studies (Zhang et al. 2005; Wang et al. 2006), TTX did not alter significantly the frequency and amplitude of spontaneous IPSCs of lamina II neurons. This is probably due to the quiescent state of GABAergic and glycinergic interneurons in the spinal cord slice. Also, the thin tissue slice preparation may compromise some of the intrinsic connectivity between individual neurons in the spinal cord in vivo.
Baclofen-induced antinociception is, in part, due to inhibition of glutamate release from primary afferents (Iyadomi et al. 2000). However, little is known about the function of presynaptic GABAB receptors in the spinal cord in diabetic neuropathy. GABAB receptors are densely located in the superficial lamina of the spinal cord, especially the primary afferent terminals (Price et al. 1984, 1987). Thus, to assess the function of GABAB receptors at the primary afferent terminals, we determined the effect of baclofen on monosynaptic EPSCs evoked from the dorsal root in control and diabetic rats. We found that baclofen significantly decreased the amplitude of eEPSCs in control and diabetic rats in a dose-dependent manner. Furthermore, the inhibitory effect of baclofen on the amplitude of monosynaptic eEPSCs was much greater in control than in diabetic rats. GABAB receptors may be preferentially expressed on C- than A
-fibre afferent terminals because baclofen differentially affects C- and A-fibre-evoked monosynaptic EPSCs in the spinal cord (Ataka et al. 2000). Since we used a thin slice preparation with a short dorsal root attached, it was not possible to distinguish between A- and C-fibre-evoked EPSCs. Based on the stimulation intensity used in our study, it is likely that both A- and C-fibre afferents were stimulated. Importantly, in both stimulation protocols (using either fixed stimulation intensity or a predetermined amplitude of eEPSCs), we consistently found that the inhibitory effect of baclofen on the amplitude of monosynaptic eEPSCs was significantly greater in control than in diabetic rats. The effect of baclofen on eEPSCs in both control and diabetic groups was completely blocked by CGP55845, a specific GABAB receptor antagonist. Additionally, we observed that the degree of inhibition on the frequency of mEPSCs by baclofen was significantly greater in the control than in the diabetic group. Hence, the above data strongly suggest that the function of GABAB receptors on the primary afferent terminals is attenuated in diabetic neuropathy. The function of GABAB receptors is critically determined by the subunits, particularly the GABAB(1A) subunit (Towers et al. 2000; Gassmann et al. 2004; Vigot et al. 2006). Diabetes may particularly affect the expression or trafficking of the GABAB(1A) subunit present on the dorsal root ganglion neurons and glutamatergic primary afferent terminals (Towers et al. 2000; Vigot et al. 2006).
It should be acknowledged that the microcircuitry of the spinal lamina II is very complex. Both excitatory and inhibitory interneurons in the lamina II are probably included in our recording. It is therefore difficult to determine if an increased excitatory input is pronociceptive or antinociceptive in this spinal slice study. Nevertheless, baclofen produced a consistent inhibitory effect on glutamatergic input in all lamina II neurons tested. This inhibitory effect on synaptic glutamate release is in agreement with the antinociceptive effect of baclofen in vivo (Malcangio & Bowery, 1993; Smith et al. 1994; Dirig & Yaksh, 1995). Also, increased glutamatergic input to lamina II neurons in diabetic rats appears to coincide with the increased hypersensitivity of spinal dorsal horn projection neurons in this rat model of diabetic neuropathic pain (Chen & Pan, 2002). Thus, it seems that increased glutamatergic input from primary afferents to lamina II neurons is largely nociceptive. Also, it should be noted that in addition to the presynaptic site, baclofen also produces a postsynaptic effect when administered in vivo. The present study only focused on presynaptic GABAB receptors, and for this reason, the postsynaptic GABAB receptors were blocked in the recorded neuron by including GDP--S.
GABAB receptors have an important role in the feedback regulation of GABAergic and glycinergic synaptic transmission in the spinal cord (Iyadomi et al. 2000; Wang et al. 2006). The function of GABAB receptors in the regulation of inhibitory input to spinal dorsal horn neurons has not been studied in diabetic neuropathic pain. In the present study, we observed that baclofen produced a profound but similar decrease in the frequency of GABAergic/glycinergic spontaneous IPSCs in control and diabetic rats. Furthermore, baclofen had a similar inhibitory effect on the frequency of GABAergic and glycinergic mIPSCs in both groups. Therefore, it appears that the presynaptic GABAB receptors present on GABAergic and glycinergic interneurons and their terminals are not significantly altered in diabetic neuropathy. Changes in GABAB receptor expression have been studied in nerve ligation-induced neuropathic pain. In this regard, sciatic axotomy causes a large reduction in GABAB mRNA levels in the spinal cord (Castro-Lopes et al. 1995; Yang et al. 2004). However, the immunoreactivity of GABAB receptors is not significantly altered in the spinal cord following ligation of the L5 spinal nerve in rats (Engle et al. 2006). Furthermore, although the antinociceptive effect of baclofen is increased, there is no significant difference in the density and binding affinity of spinal GABAB receptors in rats subjected to nerve ligation (Smith et al. 1994). Findings from the present study provide new information that the function of presynaptic GABAB receptors on the central terminals of primary afferents, but not those on GABAergic/glycinergic interneurons, is reduced in diabetic neuropathy.
In summary, our study provides important evidence that glutamatergic input from primary afferents to dorsal horn neurons is increased in diabetic neuropathic pain. Furthermore, the electrophysiological data in this rat model of diabetes suggest that GABAB receptor function at the primary afferent terminals is reduced. On the other hand, presynaptic GABAB receptors present on GABAergic and glycinergic interneurons are not significantly altered in diabetic neuropathy. Since GABAB receptors at the glutamatergic terminals can limit synaptic glutamate release in the spinal cord (Iyadomi et al. 2000; Li et al. 2002), the reduced sensitivity of GABAB receptor function in diabetic rats may contribute to increased glutamatergic input, hyperactivity of dorsal horn neurons, and development of diabetic neuropathic pain. This new information is important for our understanding of the synaptic plasticity and mechanisms responsible for central sensitization and neuropathic pain in diabetes. It has been reported that a higher dose of baclofen is required to reduce diabetic neuropathic pain (Malcangio & Tomlinson, 1998). Therefore, reduced GABAB receptor sensitivity at the primary afferent terminals in the spinal cord is probably responsible for the attenuated efficacy of the GABAB receptor agonist in diabetic neuropathic pain.
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