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NMDA receptor 2B subunit-mediated synaptic transmission in the superficial dorsal horn of peripheral nerve-injured neuropathic mice
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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s
Research Report
NMDA receptor 2B subunit-mediated synaptic transmission in the superficial dorsal horn of peripheral nerve-injured neuropathic mice Hideaki Iwata, Toshifumi Takasusuki, Shigeki Yamaguchi, Yuuichi Hori⁎ Department of Physiology and Biological Information, Dokkyo Medical University School of Medicine, Kitakobayashi 880, Mibu, Tochigi 321-0293, Japan
A R T I C LE I N FO
AB S T R A C T
Article history:
Previous research has shown that peripheral inflammation and peripheral nerve injury
Accepted 7 December 2006
alter the properties of NMDA receptors in the spinal dorsal horn. However, there is no
Available online 2 January 2007
direct evidence that demonstrates the influence of peripheral nerve injury on NMDA receptor-mediated synaptic transmission in the spinal dorsal horn. Using whole cell tight-
Keywords:
seal methods, NMDA receptor-mediated excitatory postsynaptic currents (NMDA EPSCs)
NMDA receptor
were recorded from superficial dorsal horn neurons in adult mouse spinal cord slices.
NR2B subunit
Peripheral nerve injury-induced changes in the pharmacological and electrophysiological
Neuropathic pain
properties of synaptic NMDA receptors were studied. The ratio of the amplitude of NMDA
Spinal cord
EPSCs to that of non-NMDA EPSCs was larger in nerve-ligated neuropathic mice than in
Dorsal horn neuron
sham-operated control mice. The decay phase of the NMDA EPSCs was slower in nerve-
Single-cell RT-PCR
ligated neuropathic mice. The NR2B subunit-specific NMDA receptor antagonist ifenprodil (10 μM) reduced the amplitude of the NMDA EPSCs and shortened their decay phase. The sensitivity of NMDA EPSCs to ifenprodil was significantly larger in nerve-ligated neuropathic mice than in sham-operated control mice. Single-cell RT-PCR analysis performed on superficial dorsal horn neurons showed that the incidence of NR2A mRNA-expressing neurons was reduced in nerve-ligated neuropathic mice. This result, together with the electrophysiological findings, suggests that the subunit composition of the subsynaptic NMDA receptors in the superficial dorsal horn was altered by peripheral nerve injury. Pharmacological and electrophysiological changes observed in the present experiments might be the underlying causes of the hyperalgesia and allodynia induced by peripheral nerve injury and inflammation. © 2006 Elsevier B.V. All rights reserved.
1.
Introduction
The N-methyl-D-aspartate (NMDA) subtype of glutamate receptor plays an important role in synaptic transmission, neuronal plasticity, and excitotoxic neuronal damage in the
central nervous system (Dingledine et al., 1999). The NMDA receptors located in the spinal cord have attracted particular attention because of their involvement in nociceptive modulation. Peripheral nerve injury often induces an abnormal pain state, so-called neuropathic pain, including hyperalgesia
⁎ Corresponding author. Fax: +81 282 86 0439. E-mail address: [email protected] (Y. Hori). 0006-8993/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2006.12.014
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(lowered threshold to induced pain) and allodynia (pain induced via an innocuous stimulus) (Boyce et al., 1999; Dubner and Ruda, 1992; Woolf and Thompson, 1991). The development and maintenance of these abnormal nociceptive states are known to be mediated by the activation of NMDA receptors in the spinal cord dorsal horn (Bennett et al., 2000; Boyce et al., 1999; Gao et al., 2005; Woolf and Thompson, 1991; Zeltser et al., 1991). The NMDA receptor is a hetero-oligomeric complex of several NMDA receptor subunits (NR1, NR2A-D, and NR3A-B). The subunit composition determines the pharmacological and physiological properties of the NMDA receptors (Monyer et al., 1992; Seeburg, 1993). The NR1 subunits are ubiquitously distributed in all laminae of the spinal cord. However, the distribution of the NR2 subunits in the spinal cord is controversial as differing observations have been reported (Boyce et al., 1999; Stegena and Kalb, 2001; Watanabe et al., 1992). Stegena and Kalb (2001) detected NR2A in lamina II in adult rat spinal cord, whereas NR2B, NR2C, and NR2D were undetectable. Watanabe et al. (1992) reported that NR2A was present in all parts of the grey matter apart from lamina II, and that NR2B was largely restricted to lamina II. Boyce et al. (1999) reported that the NR2B subunit had a restricted distribution in the superficial dorsal horn. Several researchers have suggested that NR2B subunits distributed in the spinal cord play an important role in neuropathic pain (Boyce et al., 1999; Chizh et al., 2001; Sakurada et al., 1998; Tan et al., 2005; Taniguchi et al.,
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1997). For example, Tan et al. (2005), reported that intrathecal administration of small interfering RNAs against the NR2B subunit suppresses the expression of NR2B in the spinal cord and reduces the pain response induced by peripheral inflammation. The functional properties of the NMDA receptor in spinal dorsal horn neurons change during neuropathic pain induced by peripheral nerve injury (Guo and Huang, 2001; Isaev et al., 2000; Karlsson et al., 2002). For example, it has been reported that in neuropathic rats, NMDA-induced whole cell membrane currents and calcium influx through NMDA receptor channels are facilitated by spinal nerve ligation (Isaev et al., 2000). In addition, the current–voltage relationship of NMDAinduced whole cell currents in dissociated rat dorsal horn neurons shifts in a hyperpolarized direction (Guo and Huang, 2001). On the other hand, Karlsson et al. (2002) reported that peripheral nerve injury and inflammation induce only a subtle change in the dose–response curves for glutamateevoked NMDA receptor-mediated whole cell currents in dissociated dorsal horn neurons. Thus, the functional properties of synaptic NMDA receptors in the spinal cord dorsal horn after peripheral nerve ligation or tissue inflammation have not been fully elucidated. In the present experiments, we recorded NMDA receptormediated excitatory postsynaptic currents (NMDA EPSCs) from the superficial dorsal horn neurons in adult mouse spinal slices, and investigated the manner in which the
Fig. 1 – Mechanical allodynia induced by the partial ligation of the sciatic nerve. (A) Mechanical withdrawal thresholds of the hindpaw ipsilateral to the sciatic nerve ligation, expressed as applied force (grams), are plotted as a function of postoperative days. Filled circles represent sciatic nerve-ligated mice (n = 35), and open circles represent sham-operated control mice (n = 24). Data are expressed as the mean ± SEM. ** Indicates a significant decrease in the withdrawal threshold compared with the values before the surgical procedure. (B) Effects of intrathecal administration of ifenprodil on the mechanical allodynia 7–10 days after nerve ligation. An open bar represents the withdrawal thresholds in sham-operated control mice 7–10 days after the surgery (n = 12). A filled bar represents the withdrawal thresholds in nerve-ligated mice that received intrathecal injection of the vehicle (1% of DMSO in saline, 5 μl, n = 9). A hatched bar represents the withdrawal thresholds in nerve-ligated mice that received intrathecal injection of ifenprodil (n = 10). ** Indicates a significant difference between sham-operated control mice and vehicle-injected nerve-lilgated mice at p < 0.01. * Indicates a significant difference between vehicle-injected nerve-ligated mice and ifenprodil-injected nerve-ligated mice at p < 0.05.
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functional properties of NMDA EPSCs are affected during allodynia induced by partial sciatic nerve ligation. By means of single-cell reverse transcription-polymerase chain reaction (RT-PCR), we also explored the manner in which partial sciatic nerve ligation affected the expression pattern of the NMDA receptor subunits in the superficial dorsal horn neurons.
2.
(2000 pmol), an NR2B receptor subunit-selective NMDA receptor antagonist, significantly increased mechanical withdrawal thresholds (Fig. 1B, a hatched bar, * indicates a significant difference compared with vehicle-injected neuropathic mice at p < 0.05). Nerve-ligated neuropathic mice that were injected with only the vehicle, showed a significant reduction in the withdrawal thresholds (Fig. 1B, a filled bar, ** indicates a significant difference compared with shamoperated control mice at p < 0.01).
Results
2.1. Behavioral assessment of mechanical allodynia induced by partial ligation of the sciatic nerve
2.2. Pharmacological isolation of NMDA and non-NMDA receptor-mediated EPSCs in the superficial dorsal horn neurons
Mechanical withdrawal thresholds of the paw ipsilateral to the sciatic nerve ligation were determined using von Frey filaments. Partial sciatic nerve ligation elicited a significant decrease in the threshold for evoking hindpaw withdrawal (Fig. 1A, filled circles, ** indicates a significant decrease in thresholds compared with the values before nerve ligation at p < 0.05). The decrease in withdrawal thresholds appeared within 1 day after nerve ligation, and lasted throughout the period of investigation up to 27 days after nerve ligation. In sham-operated control mice, no significant alterations in withdrawal thresholds were observed (Fig. 1A, open circles). The decrease in withdrawal thresholds reached a stable low level 7 to 10 days after nerve ligation (Fig. 1A, filled circles). During this period, intrathecal administration of ifenprodil
Fig. 2A shows representative examples of EPSCs recorded in a sham-operated control (left panel) and a nerve-ligated neuropathic mouse (right panel). Electrical stimulation applied via a glass pipette placed in the dorsolateral margin of the spinal cord, 50–200 μm away from the recorded neuron, evoked EPSCs in the presence of bicuculline (20 μM), strychnine (1 μM), and glycine (5 μM). The EPSCs were directed inward at a holding potential of −70 mV (Fig. 2A, a). At a depolarized holding potential of +60 mV, the direction of the EPSCs reversed to outward. Adding CNQX (10 μM), a non-NMDA receptor antagonist, abolished an early component leaving a late component (Fig. 2A,b). The late component of the EPSC was blocked by the application of APV, an NMDA receptor antagonist (50 μM, Fig. 2A,c).
Fig. 2 – NMDA and non-NMDA components of EPSCs in sham-operated and nerve-ligated mice. (A) Representative recordings of the evoked EPSCs in a sham-operated control mouse (left panel) and a nerve-ligated neuropathic mouse (right panel). The traces labeled (a) were recorded at a holding potential of −70 mV in the presence of bicuculline (10 μM), strychnine (1 μM), glycine (2 μM), and APV (10 μM). The traces labeled (b) were recorded at a holding potential of + 50 mV in the presence of bicuculline, strychinine, glycine, and CNQX (10 μM). The traces labeled (c) were recorded at a holding potential of +50 mV in the presence of bicuculline, strychinine, glycine, APV, and CNQX. (B) Bar graph summarizing the ratio of NMDA receptor-mediated component of EPSC to the non-NMDA receptor-mediated component of EPSC recorded in sham-operated control mice (n = 12) and nerve-ligated neuropathic mice (n = 15). Bars and vertical lines indicate the mean values ± SEM. * Indicates significant difference at p < 0.05 (unpaired t-test).
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2.3. NMDA/non-NMDA ratio of the synaptic responses in control and neuropathic mice
2.4. The decay time course of the NMDA EPSC in control and neuropathic mice
The contribution of the NMDA receptor to the total postsynaptic current in sham-operated control mice was compared with that in nerve-ligated neuropathic mice. The NMDA EPSC was recorded at +60 mV in the presence of bicuculline, strychnine, glycine, and CNQX, and its amplitude was measured 75 ms after stimulation. The non-NMDA EPSC was recorded at −70 mV in the presence of bicuculline, strychnine, glycine, and APV, and its amplitude was measured at its peak. The ratio of the amplitude of NMDA EPSC to that of non-NMDA EPSC in nerve-ligated neuropathic mice was 2.18 ± 0.103 (mean ± SEM, n = 20), which was significantly larger than that in sham-operated control mice (0.817 ± 0.014, n = 12, unpaired t-test, p < 0.001, Fig. 2B).
The left trace in Fig. 3A shows the NMDA EPSC in a shamoperated control mouse along with double exponential fits to the decay phase. The time constants of the fast (τ1) and slow (τ2) components were 21.8 ms and 180.5 ms, respectively. The contribution of the slow component to total synaptic current was estimated to be 28%. The right trace in Fig. 3A shows NMDA EPSC in a nerveligated neuropathic mouse, together with double exponential fits to the decay phase. τ1 and τ2 were 63.5 ms and 299.8 ms, respectively. The contribution of the slow component to the total synaptic current was estimated to be 42%. The NMDA EPSCs recorded from 17 neurons in nerveligated neuropathic mice and those from 10 neurons in sham-
Fig. 3 – Decay kinetics of NMDA EPSCs in sham-operated and nerve-ligated mice. (A) Representative recordings of evoked EPSCs in a sham-operated control mouse (left panel) and a nerve-ligated neuropathic mouse (right panel). Recordings were made at a holding potential of +50 mV in the presence of bicuculline, strychnine, glycine, and CNQX. In both cases, the decay was best fitted by a double exponential function. The fitted functions are superimposed (thin line) with their time constants (slow and fast components of NMDA EPSCs). The number in parentheses represents the amplitude of the slow component relative to the total amplitude of the NMDA EPSCs. (B) Bar graphs summarizing decay kinetics of NMDA EPSCs in sham-operated control mice (shadowed bars) and nerve-ligated neuropathic mice (open bars). The left panel shows the time constant of the fast component (τ1), the middle panel shows the slow component (τ2), and the right panel shows the relative amplitude of the slow component. Bars and vertical lines indicate the mean values ± SEM. * Indicates significance at p < 0.05. ** Indicates significance at p < 0.01. NS indicates “not significant.”
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Fig. 4 – Effects of ifenprodil on NMDA EPSCs in nerve-ligated mice. (A) Representative recordings of the NMDA EPSCs evoked before (left panel) and during (right panel) the application of ifenprodil. The recordings were made at a holding potential of +50 mV in the presence of bicuculline, strychnine, glycine, and CNQX. Ifenprodil was applied by bath perfusion at a concentration of 10 μM. The decay was best fitted by a double exponential function in both cases. The fitted functions are superimposed (thin line) with their time constants (slow and fast components of NMDA EPSCs). The number in parentheses represents the relative amplitude of the slow component of the NMDA EPSCs. (B) Graphs summarizing the effects of ifenprodil on NMDA EPSCs in sham-operated control mice (filled circles) and nerve-ligated neuropathic mice (open circles). The leftmost graph shows the effect of ifenprodil on the ratio of the NMDA EPSC amplitude to the non-NMDA EPSC amplitude. The second and third graphs from the left show the effect of ifenprodil on the time constant of the fast (τ1) and slow (τ2) components of NMDA EPSCs, respectively. The rightmost graph shows its effect on the relative amplitude of the slow component of NMDA EPSCs. Symbols and vertical lines indicate the mean values ± SEM. * Indicates significance at p < 0.05. ** Indicates significance at p < 0.01. NS indicates “not significant.”
operated control mice were fitted with a double exponential function. τ1 and τ2 were 31.4 ± 6.7 ms and 201.5 ± 10.7 ms (mean ± SEM), respectively, in sham-operated control mice (Fig. 3B, leftmost and middle graphs, shadowed bars). τ1 and τ2 in nerve-ligated neuropathic mice were 68.5 ± 9.3 ms and 312.8 ± 19.5 ms, respectively (Fig. 3B, leftmost and middle graphs, open bars). τ2 in nerve-ligated neuropathic mice was statistically longer than those in sham-operated control mice (p < 0.01). The relative contribution of the slow component of NMDA EPSCs was 46.0 ± 7.2% in nerve-ligated neuropathic mice, which is significantly larger than that in sham-operated control mice (29.1 ± 8.7%, p < 0.05, Fig. 3B, graph at the extreme right).
2.5.
neurons in sham-operated control mice. Fig. 4 is an example of the agarose gel electrophoresis of the RT-PCR products obtained by single-cell RT-PCR analysis. Lanes labeled cell #1 are for a neuron from a sham-operated control mouse, and lanes labeled cell #2 are for a neuron from a nerve-ligated neuropathic mouse. The PCR product of neuron-specific enolase (NSE) mRNA was detected in all the cells examined. Of the 51 neurons in nerve-ligated mice, 15 expressed NR2A mRNA but not NR2B mRNA, 12 neurons expressed both NR2A and NR2B, and 16 neurons expressed NR2B but not NR2A; NR2A-B subunits were not detected in 8 neurons (Table 1, upper row). Of the 49 neurons in sham-operated control mice, 32 neurons expressed NR2A but not NR2B, 9 neurons expressed both NR2A and NR2B, and 2 neurons expressed NR2B but not NR2A; NR2A-B subunits were not detected in 6 neurons (Table 1, lower row). The Chi-square test indicates that the incidence of NR2A subunit expression was significantly larger in sham-operated control mice than in nerve-ligated neuropathic mice (p < 0.01). Further, the incidence of NR2B subunit expression was significantly larger in nerve-ligated neuropathic mice than in sham-operated control mice (p < 0.01).
2.6.
Effects of ifenprodil on NMDA receptor-mediated EPSC
Fig. 5A shows the effects of ifenprodil, an NR2B antagonist, at a concentration of 10 μM on an NMDA EPSC in a nerve-ligated neuropathic mouse. Ifenprodil slightly but distinctly decreased the amplitude of NMDA EPSC in this example. Fitting the double exponential function to the decay time course indicated that ifenprodil shortened the decay phase and decreased the relative contribution of the slow component to the EPSC. The effects of ifenprodil were studied in 8 neurons in nerve-ligated neuropathic mice and in 8 neurons in shamoperated control mice. Grouped data are shown in Fig. 5B, which indicates that nerve-ligated neuropathic and shamoperated control mice have significantly different susceptibilities to ifenprodil [two-way analysis of variance (ANOVA), p < 0.05]. In nerve-ligated neuropathic mice (Fig. 5B, open circles), ifenprodil decreased the amplitude of NMDA EPSC to 34.3 ± 5.1% of the control (p < 0.05); the time constant of the fast component, from 52.6 ± 7.8 ms to 39.2 ± 3.5 ms (no significant difference); the time constant of the slow component, from 302.3 ± 24.7 ms to 181.0 ± 23.2 ms (p < 0.01); and the relative contribution of the slow component to the synaptic current, from 42.9 ± 5.9% to 21.2 ± 8.1% (p < 0.05). Single-cell RT-PCR
Table 1 – Expression pattern of NMDA receptor subunits in sham-operated and nerve-ligated mice NR2A
NR2B
NR2A and NR2B
None
Total number of neurons
15
16
12
8
51
32
2
9
6
49
Single-cell RT-PCR analysis of NMDA subunits
After patch clamp recordings, we collected the neuron under investigation. Single-cell RT-PCR analysis was performed on 51 neurons in nerve-ligated neuropathic mice and on 49
Nerve-ligated mice Sham-operated mice
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Fig. 5 – Single-cell RT-PCR analysis of NMDA receptor subunit expression in sham-operated and nerve-ligated mice. Lanes labeled cell #1 are for a neuron sampled from a sham-operated control mouse. Lanes labeled cell #2 are for a neuron sampled from a nerve-ligated neuropathic mouse. The lane labeled “Marker” contains the 100-bp DNA ladder. Values in parentheses on the left indicate the expected length of the PCR products for NSE, NR2A, and NR2B.
analysis revealed that 4 of these 8 neurons expressed only NR2B mRNA, 3 of these 8 neurons expressed both NR2AB and NR2B mRNAs, and no RT-PCR product was detected in the remaining neuron. In sham-operated control mice (Fig. 5B, filled circles), ifenprodil decreased the amplitude of NMDA EPSC to 57.4% ± 11.4% of the control (p < 0.05). However, the time constants of the fast and slow components or the relative contribution of the slow component were not significantly altered by ifenprodil (p > 0.05). Single-cell RT-PCR analysis showed that 6 of these 8 neurons expressed only NR2A mRNA, 2 of these 8 neurons expressed both NR2A and NR2B mRNAs. The differential expression patterns of NMDA receptor subunits between nerve-ligated neuropathic mice and sham-operated control mice are consistent with the observed differential effects of ifenprodil.
3.
Discussion
In the present experiments, we recorded synaptically evoked NMDA EPSCs from the superficial dorsal horn neurons in the spinal cord slice. The functional properties of the NMDA
receptors expressed in the synaptic regions of nerve-ligated neuropathic mice were compared with those of shamoperated control mice.
3.1. The functional properties of NMDA EPSCs changed in nerve-injured mice The glutamatergic EPSCs evoked by the electrical stimulation of adjacent fibers were recorded from neurons located in the superficial dorsal horn. The glutamatergic EPSCs were composed of two components, NMDA EPSC and nonNMDA EPSC. The ratio of the amplitude of the NMDA EPSC to that of the non-NMDA EPSC was significantly larger in nerveligated neuropathic mice than in sham-operated control mice. Although the fast and slow time constants of NMDA EPSCs recorded in sham-operated control mice varied considerably among neurons, they are in the range of the previously reported values in rat dorsal horn neurons which express NR2A subunit-containing NMDA receptors (Bardoni et al., 1998; Momiyama, 2000). The results also showed that the NMDA EPSCs had slower decay time courses in nerve-ligated neuropathic mice than in sham-operated control mice.
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A detailed analysis of the peripheral nerve injury-induced alterations of the properties of the NMDA receptors in the spinal cord dorsal horn was previously reported (Guo and Huang, 2001; Isaev et al., 2000; Karlsson et al., 2002). For example, Guo and Huang made patch clamp recordings from isolated spinal dorsal horn neurons and analyzed whole cell currents that were induced by pressure-applied NMDA. Peripheral inflammation induced by injections of complete Freund's adjuvant reduced the magnesium block of NMDA responses and shifted the voltage dependence of the NMDA receptors toward the hyperpolarized direction (Guo and Huang, 2001). Karlsson et al. recorded NMDA receptor-mediated currents evoked by glutamate application in single neurons dissociated from the rat spinal cord. They reported that despite minor changes in the dose–response relation of glutamate-evoked NMDA receptor-mediated currents after the nerve lesion, there were no significant differences in the density and magnesium block of the current between the sham-operated control and nerve-ligated neuropathic rats (Karlsson et al., 2002). Isaev et al. recorded whole cell membrane currents and calcium transient in superficial dorsal horn neurons in the rat spinal cord slice preparation. They found that spinal nerve ligation produces prolonged facilitation of membrane currents and calcium transient induced by bath application of NMDA. They suggested that the increase in calcium signaling mediated by NMDA receptors is involved in the development of neuropathic pain following spinal nerve injury (Isaev et al., 2000). The present results are in line with this previous report and provide direct evidence that the relative contribution of the NMDA receptor to excitatory synaptic transmission in the superficial dorsal horn increases with peripheral nerve injury.
3.2. Effects of the NR2B subunit-specific antagonist ifenprodil on NMDA EPSCs in nerve-ligated neuropathic mice The NR2B subunit-specific NMDA receptor antagonist ifenprodil (10 μM) reduced the amplitude of the NMDA EPSCs and shortened their decay phase. These effects of ifenprodil were more prominent in nerve-ligated neuropathic mice than in sham-operated control mice. Momiyama investigated the neurons in the superficial dorsal horn of adult rats and reported that ifenprodil (10 μM) reduced whole cell current induced by NMDA application, but ifenprodil did not affect synaptically evoked NMDA EPSCs. In contrast to this, we observed that ifenprodil (10 μM) reduced the amplitude of the NMDA EPSCs. The contrasting results of ifenprodil's effects on synaptically evoked NMDA EPSCs in the spinal cord dorsal horn, although not fully clear, may be partially explained by the differences in stimulation methods. In this study, NMDA EPSCs were evoked by a glass pipette placed near the recorded neurons, while Momiyama stimulated a dorsal root by a suction electrode. Dorsal root stimulation and intraspinal stimulation may activate different groups of synapses with different subunit composition, or they may activate the NMDA receptors to different extents. It has been suggested that the effects of ifenprodil depend on the extent of NMDA receptor activation (Kew et al., 1996; Zhang et al., 2000). At low NMDA concentration, ifenprodil enhances
NMDA-induced currents, while at high NMDA concentration, the NMDA-induced currents are inhibited. Nagy et al. (2004) analyzed the synaptic distribution of the NR2B subunit in the rat lumbar spinal cord by using an antigen-unmasking technique. They demonstrated that the NR2B subunit is concentrated in the superficial dorsal horn, and suggested that there are high levels of NR2B in many synaptic regions. Our observation that ifenprodil suppressed synaptically evoked NMDA EPSCs seems consistent with this report.
3.3. Single-cell RT-PCR analysis of NMDA receptor subunits expressed in the superficial dorsal horn The previously reported single-cell RT-PCR analysis on neurons dissociated from the spinal cord dorsal horn showed that fewer neurons express the NR2A in peripheral nerveinjured rats as compared to those in the control rats, and the incidence of neurons expressing the NR2B increased proportionally in nerve-injured rats (Karlsson et al., 2002). It has also been reported that, in mice, the expression level of NR2A in the dorsal horn decreased after injection of formalin into the hindpaw (Gaunitz et al., 2002). Furthermore, exaggerated nociceptive states induced by peripheral nerve injury or tissue inflammation is known to be mediated by the activation of NR2B subunits distributed in the spinal cord dorsal horn (Boyce et al., 1999; Chizh et al., 2001; Sakurada et al., 1998; Tan et al., 2005; Taniguchi et al., 1997). These previous reports prompted us to compare the expression pattern of NR2A and NR2B in the superficial dorsal horn neurons between shamoperated control mice and nerve-ligated neuropathic mice. The results showed that the ratio of the number of neurons expressing NR2A subunit decreased, whereas the ratio of the number of neurons expressing NR2B subunit increased after nerve ligation. This observation is consistent with the abovementioned report of single-cell RT-PCR analysis on dissociated spinal cord neurons of neuropathic rats.
3.4. Possible mechanisms for the observed alterations in the functional properties of NMDA receptors in nerve-ligated neuropathic pain Substantial evidence indicates that the neuropathic pain induced by peripheral nerve injury is accompanied by alterations in synaptic transmission in the spinal cord dorsal horn. The alterations associated with neuropathic pain include phosphorylation of NMDA receptors (Guo et al., 2002; Zou et al., 2002), altered NMDA receptor subunit expression pattern (Gaunitz et al., 2002; Karlsson et al., 2002), conversion of silent synapses containing only NMDA receptors to functional synapses (Baba et al., 2000), and an increase in NMDAmediated current (Isaev et al., 2000). Although the precise mechanisms for the presently observed alterations in the functional properties of NMDA EPSCs in nerve-ligated neuropathic mice are not clear, our eletrophysiological observations, together with single-cell RT-PCR analysis appear to indicate that the subunit composition of the NMDA receptors expressed in the synaptic region might change after nerve injury. The results of single-cell RT-PCR analysis show that the incidence of NR2A decreased and that of NR2B increased after
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peripheral nerve injury leading to a slower decay time course and a higher susceptibility to ifenprodil, as observed in the nerve-ligated neuropathic mice. The NR2 subunits are known to determine the kinetic properties of the NMDA receptors (Cathala et al., 2000; Misra et al., 2000; Vicini et al., 1998; Wyllie et al., 1998). Increased NR2A subunit expression and decreased NR2B subunit expression accelerate NMDA EPSCs (Cathala et al., 2000; Cull-Candy and Leszkiewicz, 2004; Flint et al., 1997). Developmental changes in the subunit composition of the NMDA receptors have been reported. During postnatal development, insertion of the NR2A subunit and loss of the NR2B subunit shorten the decay time course of the NMDA EPSCs (Cathala et al., 2000). Furthermore, even after neuronal development, the NMDA receptor subunit expression pattern was reported to change in an activity-dependent manner. For example, it has been shown that suppressing neuronal activity results in the re-expression of NR2B subunits (Ehlers, 2003; Kiyosue et al., 2004). It is conceivable that alterations in synaptic activity resulting from peripheral nerve ligation might alter the NMDA receptor subunit expression pattern in the spinal cord dorsal horn. Finally, NR1 and NR2A subunits are also reported to be involved in neuropathic pain. Further studies on these NMDA receptor subunits would provide clues for further understanding the cellular mechanisms of neuropathic pain induced by peripheral nerve injury.
In some mice, ifenprodil (2000 pmol, Tan-No et al., 2000), a selective antagonist against the NMDA receptor 2B subunit, was administered intrathecally between lumbar segment L5 and L6 according to the procedure described by Hylden and Wilcox (1980) under unanesthetized conditions. The intrathecal location of the needle tip was affirmed by a characteristic flick of the tail. The withdrawal thresholds were measured 10– 20 min after the intrathecal administration of ifenprodil. Ifenprodil (RBI, MA, U.S.A.) was dissolved in 1% dimethyl sulphoxide (DMSO) in saline. Some nerve-ligated mice were injected with the vehicle only. The intrathecal administration and threshold measurements were performed 7 (n = 12), 8 (n = 12), 9 (n = 10), and 10 (n = 10) days after the partial nerve ligation, and all the data were pooled for further analysis.
4.4.
Experimental procedures
4.1.
Animals
Experiments were conducted using adult male ICR mice (7–8 weeks old) that were kept at controlled room temperature under a 12-h light/dark cycle. The care and use of the animals were in accordance with institutional guidelines and the guidelines of the International Association for the Study of Pain (Zimmermann, 1983).
4.2.
Surgery
The mice were anesthetized by an intraperitoneal injection of sodium pentobarbital (50 mg/kg). Partial ligation of the left sciatic nerve was made according to the protocol for rats described by Seltzer et al. (1990). In sham-operated control mice, the sciatic nerve was exposed but not ligated.
4.3.
Assessment of behavioral mechanical
Mice were placed on an elevated plastic mesh floor covered with a clear plastic box (height, 15 cm; diameter, 12 cm). The withdrawal threshold to mechanical stimulation was determined. The mechanical stimulus was applied from underneath to the plantar aspect of the hindlimb, ipsilateral to the partial nerve ligation by means of an Elctro von Frey (Model 1601; IITC Inc., Woodland Hills, CA). The lowest force from five tests that induced a withdrawal response was considered the withdrawal threshold. The measurements were made from 2 days before the surgical operation through 27 days after the surgical operation.
Preparation of spinal cord slices
Transverse slices were obtained 7–10 days after sciatic nerve ligation. Segments of the lumbosacral (L4-S1) spinal cord were removed under deep ether anesthesia. A Vibratome (Dosaka EM, Japan) was used to cut transverse slices (350–450 μm) in Krebs solution at 4 °C. The Krebs solution was equilibrated with 95% O2 and 5% CO2 and contained the following (in mM): NaCl, 113; KCl, 3; NaHCO3, 25; NaH2PO4, 1; CaCl2, 2; MgCl2, 1; Dglucose, 11; pH 7.4.
4.5.
4.
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Whole cell recordings
After a 1-h incubation period in Krebs solution at 37 °C, the slices were mounted onto a recording chamber on a microscope stage and continuously perfused with Krebs solution. Conventional tight-seal whole cell recordings were made from visually identified neurons located in the superficial dorsal horn (laminae II and III) of the spinal cord slice using infrared DIC videomicroscopy as previously described (Wake et al., 2001). Since no apparent difference in the characteristics of the NMDA receptor-mediated postsynaptic currents was observed, data obtained from the neurons in laminae II and III were pooled for analysis. Patch pipettes were made from thin-wall borosilicate glass capillaries pulled on a Flaming Brown pipette puller (Sutter Instruments, Novato, CA, U.S.A.) and filled with a solution of the following composition (in mM): K gluconate, 123; KCl, 14; Na gluconate, 2; EGTA, 1; HEPES, 10; pH neutralized to 7.4 with KOH. The DC resistance of the pipettes filled with the internal solution was 5–10 MΩ. The membrane potential value was corrected for junction potential. Fast and slow capacitances were neutralized. The series resistance was compensated by 60%. The access resistance (11–20 MΩ) was continuously monitored and data were discarded when the value changed by more than 10%. EPSCs were evoked at 0.1 Hz by using a stimulating electrode filled with 1 M NaCl with its tip (diameter, ca. 3 μm) placed at the dorsolateral margin of the spinal cord, 100– 200 μm away from the recording site. All recordings were made in the presence of strychnine (Sigma, 0.5–1.0 μM), bicuculline (Sigma, 20 μM), and glycine (Sigma, 5 μM). To record the non-NMDA receptor-mediated component of EPSCs (non-NMDA EPSCs), each neuron was held at −70 mV in the presence of AP5 (50 μM). To record the NMDA receptor-
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mediated component of EPSCs (NMDA EPSCs), each neuron was held at +60 mV in the presence of CNQX (10 μM). EPSCs were recorded in the voltage clamp mode using an Axopatch 200B patch clamp amplifier (Axon Instruments). Data were sampled at a rate of 10.0 kHz through a Digidata 1230 interface (Axon Instruments). pCLAMP (Axon Instruments) was used to analyze the data.
4.6.
Single-cell RT-PCR
After whole cell recordings were made, the neurons were aspirated into another pipette following a previously described protocol (Tsuchiya et al., 1999). The collecting pipette had a tip diameter of about 3–5 μm and contained 2 μl of Ca2+- and Mg2+-free phosphate-buffered saline solution. The neurons were then ejected into thin-walled autoclaved PCR tubes by applying a gentle positive pressure, and immediately frozen and stored at − 80 °C until use. The PCR tubes contained 2 μl MgCl2 (25 mM), 2 μl 10 PCR buffer (200 mM Tris–HCl, 500 mM KCl), 0.5 μl RNase inhibitor (40,000 units/ml), 2 μl nonionic detergent IGEPAL CA-630 (5%), and 5 μl DEPCtreated water. On the following day, lysis was performed using IGEPAL CA-630 at room temperature for 5 min and the reversetranscription mixture was added. It contained 1 μl oligo (dT) primers (0.5 μg/μl), 2 μl mixed deoxynucleotide triphosphates (dNTPs, 10 mM), 2 μl dithiothreitol (0.1 M), 0.5 μl RNase inhibitor (40,000 units/ml), and 1 μl SuperScript II RT (200 Units/μl). The reaction mixture was incubated at 42 °C for 50 min. Subsequently, the sample was heat inactivated at 70 °C for 15 min. The PCR reactions were performed after the protocol of Fukushima et al. (2005) in a 50-μl volume containing 20 mM Tris–HCl, 50 mM KCl, 2.5 mM MgCl2, 0.2 mM dNTPs, and 2.5 units Taq DNA polymerase. The entire RT product was used for the first PCR, and 1 to 2 μl of the first PCR product was used for the second PCR. The primers used targeted three genes, NSE, and the NMDA receptor subunits NR2A and NR2B. NSE was used as a positive control. Primers used for detection of NSE were 5′-ATAGTGGGCGATGACC TGAC-3′ and 5′-ATGAACGTGTCCTCCGTTTC-3′ (200-bp product; GenBank accession # NM013509). Primers used for NR2A subunit detection were 5′-GCTACGGGCA GACAGAGAAG-3′ and 5′-GTGGTTGTCATCTGGCTCAC-3′ (257-bp product; GenBank accession # NM008170). Primers used for NR2B subunit detection were 5′-GCTACAACACC CACGAGAAGAG-3′ and 5′-GAGAGGGTCCACGCTTTCC-3′ (314-bp product; GenBank accession # NM008171). These primers were used for both the first and second PCR amplifications. Each primer was individually used in the second PCR. The concentration of primers was 20 nM each in the first PCR and 200 nM in the second PCR. The thermal cycler (Gene Amp 2400, Perkin Elmer) was programmed for 35–40 cycles of 1 min denaturation (94 °C), 1 min annealing (54–59 °C), and 1 min elongation (72 °C). The second PCR products were visualized by staining with ethidium bromide and separated by agarose gel electrophoresis. All the products were sequenced using dye terminator chemistry (Applied Biosystems) and a DNA sequencer (Model 377, ABI) and matched the published sequences.
All the reagents for the RT-PCR, except the RNase inhibitor (TOYOBO), IGEPAL CA-630 (Sigma), and Taq DNA polymerase (Takara), were obtained from GIBCO BRL.
4.7.
Statistical analysis
Results are expressed as mean ± SEM. Statistical analysis of the results was performed by one-way or two-way analysis of variance (ANOVA) followed by post hoc multiple comparison (Tukey test). Student's t-test and kai2 test were also used when appropriate. Differences were considered significant at the level of p < 0.05.
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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s
Research Report
NMDA receptor 2B subunit-mediated synaptic transmission in the superficial dorsal horn of peripheral nerve-injured neuropathic mice Hideaki Iwata, Toshifumi Takasusuki, Shigeki Yamaguchi, Yuuichi Hori⁎ Department of Physiology and Biological Information, Dokkyo Medical University School of Medicine, Kitakobayashi 880, Mibu, Tochigi 321-0293, Japan
A R T I C LE I N FO
AB S T R A C T
Article history:
Previous research has shown that peripheral inflammation and peripheral nerve injury
Accepted 7 December 2006
alter the properties of NMDA receptors in the spinal dorsal horn. However, there is no
Available online 2 January 2007
direct evidence that demonstrates the influence of peripheral nerve injury on NMDA receptor-mediated synaptic transmission in the spinal dorsal horn. Using whole cell tight-
Keywords:
seal methods, NMDA receptor-mediated excitatory postsynaptic currents (NMDA EPSCs)
NMDA receptor
were recorded from superficial dorsal horn neurons in adult mouse spinal cord slices.
NR2B subunit
Peripheral nerve injury-induced changes in the pharmacological and electrophysiological
Neuropathic pain
properties of synaptic NMDA receptors were studied. The ratio of the amplitude of NMDA
Spinal cord
EPSCs to that of non-NMDA EPSCs was larger in nerve-ligated neuropathic mice than in
Dorsal horn neuron
sham-operated control mice. The decay phase of the NMDA EPSCs was slower in nerve-
Single-cell RT-PCR
ligated neuropathic mice. The NR2B subunit-specific NMDA receptor antagonist ifenprodil (10 μM) reduced the amplitude of the NMDA EPSCs and shortened their decay phase. The sensitivity of NMDA EPSCs to ifenprodil was significantly larger in nerve-ligated neuropathic mice than in sham-operated control mice. Single-cell RT-PCR analysis performed on superficial dorsal horn neurons showed that the incidence of NR2A mRNA-expressing neurons was reduced in nerve-ligated neuropathic mice. This result, together with the electrophysiological findings, suggests that the subunit composition of the subsynaptic NMDA receptors in the superficial dorsal horn was altered by peripheral nerve injury. Pharmacological and electrophysiological changes observed in the present experiments might be the underlying causes of the hyperalgesia and allodynia induced by peripheral nerve injury and inflammation. © 2006 Elsevier B.V. All rights reserved.
1.
Introduction
The N-methyl-D-aspartate (NMDA) subtype of glutamate receptor plays an important role in synaptic transmission, neuronal plasticity, and excitotoxic neuronal damage in the
central nervous system (Dingledine et al., 1999). The NMDA receptors located in the spinal cord have attracted particular attention because of their involvement in nociceptive modulation. Peripheral nerve injury often induces an abnormal pain state, so-called neuropathic pain, including hyperalgesia
⁎ Corresponding author. Fax: +81 282 86 0439. E-mail address: [email protected] (Y. Hori). 0006-8993/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2006.12.014
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(lowered threshold to induced pain) and allodynia (pain induced via an innocuous stimulus) (Boyce et al., 1999; Dubner and Ruda, 1992; Woolf and Thompson, 1991). The development and maintenance of these abnormal nociceptive states are known to be mediated by the activation of NMDA receptors in the spinal cord dorsal horn (Bennett et al., 2000; Boyce et al., 1999; Gao et al., 2005; Woolf and Thompson, 1991; Zeltser et al., 1991). The NMDA receptor is a hetero-oligomeric complex of several NMDA receptor subunits (NR1, NR2A-D, and NR3A-B). The subunit composition determines the pharmacological and physiological properties of the NMDA receptors (Monyer et al., 1992; Seeburg, 1993). The NR1 subunits are ubiquitously distributed in all laminae of the spinal cord. However, the distribution of the NR2 subunits in the spinal cord is controversial as differing observations have been reported (Boyce et al., 1999; Stegena and Kalb, 2001; Watanabe et al., 1992). Stegena and Kalb (2001) detected NR2A in lamina II in adult rat spinal cord, whereas NR2B, NR2C, and NR2D were undetectable. Watanabe et al. (1992) reported that NR2A was present in all parts of the grey matter apart from lamina II, and that NR2B was largely restricted to lamina II. Boyce et al. (1999) reported that the NR2B subunit had a restricted distribution in the superficial dorsal horn. Several researchers have suggested that NR2B subunits distributed in the spinal cord play an important role in neuropathic pain (Boyce et al., 1999; Chizh et al., 2001; Sakurada et al., 1998; Tan et al., 2005; Taniguchi et al.,
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1997). For example, Tan et al. (2005), reported that intrathecal administration of small interfering RNAs against the NR2B subunit suppresses the expression of NR2B in the spinal cord and reduces the pain response induced by peripheral inflammation. The functional properties of the NMDA receptor in spinal dorsal horn neurons change during neuropathic pain induced by peripheral nerve injury (Guo and Huang, 2001; Isaev et al., 2000; Karlsson et al., 2002). For example, it has been reported that in neuropathic rats, NMDA-induced whole cell membrane currents and calcium influx through NMDA receptor channels are facilitated by spinal nerve ligation (Isaev et al., 2000). In addition, the current–voltage relationship of NMDAinduced whole cell currents in dissociated rat dorsal horn neurons shifts in a hyperpolarized direction (Guo and Huang, 2001). On the other hand, Karlsson et al. (2002) reported that peripheral nerve injury and inflammation induce only a subtle change in the dose–response curves for glutamateevoked NMDA receptor-mediated whole cell currents in dissociated dorsal horn neurons. Thus, the functional properties of synaptic NMDA receptors in the spinal cord dorsal horn after peripheral nerve ligation or tissue inflammation have not been fully elucidated. In the present experiments, we recorded NMDA receptormediated excitatory postsynaptic currents (NMDA EPSCs) from the superficial dorsal horn neurons in adult mouse spinal slices, and investigated the manner in which the
Fig. 1 – Mechanical allodynia induced by the partial ligation of the sciatic nerve. (A) Mechanical withdrawal thresholds of the hindpaw ipsilateral to the sciatic nerve ligation, expressed as applied force (grams), are plotted as a function of postoperative days. Filled circles represent sciatic nerve-ligated mice (n = 35), and open circles represent sham-operated control mice (n = 24). Data are expressed as the mean ± SEM. ** Indicates a significant decrease in the withdrawal threshold compared with the values before the surgical procedure. (B) Effects of intrathecal administration of ifenprodil on the mechanical allodynia 7–10 days after nerve ligation. An open bar represents the withdrawal thresholds in sham-operated control mice 7–10 days after the surgery (n = 12). A filled bar represents the withdrawal thresholds in nerve-ligated mice that received intrathecal injection of the vehicle (1% of DMSO in saline, 5 μl, n = 9). A hatched bar represents the withdrawal thresholds in nerve-ligated mice that received intrathecal injection of ifenprodil (n = 10). ** Indicates a significant difference between sham-operated control mice and vehicle-injected nerve-lilgated mice at p < 0.01. * Indicates a significant difference between vehicle-injected nerve-ligated mice and ifenprodil-injected nerve-ligated mice at p < 0.05.
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functional properties of NMDA EPSCs are affected during allodynia induced by partial sciatic nerve ligation. By means of single-cell reverse transcription-polymerase chain reaction (RT-PCR), we also explored the manner in which partial sciatic nerve ligation affected the expression pattern of the NMDA receptor subunits in the superficial dorsal horn neurons.
2.
(2000 pmol), an NR2B receptor subunit-selective NMDA receptor antagonist, significantly increased mechanical withdrawal thresholds (Fig. 1B, a hatched bar, * indicates a significant difference compared with vehicle-injected neuropathic mice at p < 0.05). Nerve-ligated neuropathic mice that were injected with only the vehicle, showed a significant reduction in the withdrawal thresholds (Fig. 1B, a filled bar, ** indicates a significant difference compared with shamoperated control mice at p < 0.01).
Results
2.1. Behavioral assessment of mechanical allodynia induced by partial ligation of the sciatic nerve
2.2. Pharmacological isolation of NMDA and non-NMDA receptor-mediated EPSCs in the superficial dorsal horn neurons
Mechanical withdrawal thresholds of the paw ipsilateral to the sciatic nerve ligation were determined using von Frey filaments. Partial sciatic nerve ligation elicited a significant decrease in the threshold for evoking hindpaw withdrawal (Fig. 1A, filled circles, ** indicates a significant decrease in thresholds compared with the values before nerve ligation at p < 0.05). The decrease in withdrawal thresholds appeared within 1 day after nerve ligation, and lasted throughout the period of investigation up to 27 days after nerve ligation. In sham-operated control mice, no significant alterations in withdrawal thresholds were observed (Fig. 1A, open circles). The decrease in withdrawal thresholds reached a stable low level 7 to 10 days after nerve ligation (Fig. 1A, filled circles). During this period, intrathecal administration of ifenprodil
Fig. 2A shows representative examples of EPSCs recorded in a sham-operated control (left panel) and a nerve-ligated neuropathic mouse (right panel). Electrical stimulation applied via a glass pipette placed in the dorsolateral margin of the spinal cord, 50–200 μm away from the recorded neuron, evoked EPSCs in the presence of bicuculline (20 μM), strychnine (1 μM), and glycine (5 μM). The EPSCs were directed inward at a holding potential of −70 mV (Fig. 2A, a). At a depolarized holding potential of +60 mV, the direction of the EPSCs reversed to outward. Adding CNQX (10 μM), a non-NMDA receptor antagonist, abolished an early component leaving a late component (Fig. 2A,b). The late component of the EPSC was blocked by the application of APV, an NMDA receptor antagonist (50 μM, Fig. 2A,c).
Fig. 2 – NMDA and non-NMDA components of EPSCs in sham-operated and nerve-ligated mice. (A) Representative recordings of the evoked EPSCs in a sham-operated control mouse (left panel) and a nerve-ligated neuropathic mouse (right panel). The traces labeled (a) were recorded at a holding potential of −70 mV in the presence of bicuculline (10 μM), strychnine (1 μM), glycine (2 μM), and APV (10 μM). The traces labeled (b) were recorded at a holding potential of + 50 mV in the presence of bicuculline, strychinine, glycine, and CNQX (10 μM). The traces labeled (c) were recorded at a holding potential of +50 mV in the presence of bicuculline, strychinine, glycine, APV, and CNQX. (B) Bar graph summarizing the ratio of NMDA receptor-mediated component of EPSC to the non-NMDA receptor-mediated component of EPSC recorded in sham-operated control mice (n = 12) and nerve-ligated neuropathic mice (n = 15). Bars and vertical lines indicate the mean values ± SEM. * Indicates significant difference at p < 0.05 (unpaired t-test).
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2.3. NMDA/non-NMDA ratio of the synaptic responses in control and neuropathic mice
2.4. The decay time course of the NMDA EPSC in control and neuropathic mice
The contribution of the NMDA receptor to the total postsynaptic current in sham-operated control mice was compared with that in nerve-ligated neuropathic mice. The NMDA EPSC was recorded at +60 mV in the presence of bicuculline, strychnine, glycine, and CNQX, and its amplitude was measured 75 ms after stimulation. The non-NMDA EPSC was recorded at −70 mV in the presence of bicuculline, strychnine, glycine, and APV, and its amplitude was measured at its peak. The ratio of the amplitude of NMDA EPSC to that of non-NMDA EPSC in nerve-ligated neuropathic mice was 2.18 ± 0.103 (mean ± SEM, n = 20), which was significantly larger than that in sham-operated control mice (0.817 ± 0.014, n = 12, unpaired t-test, p < 0.001, Fig. 2B).
The left trace in Fig. 3A shows the NMDA EPSC in a shamoperated control mouse along with double exponential fits to the decay phase. The time constants of the fast (τ1) and slow (τ2) components were 21.8 ms and 180.5 ms, respectively. The contribution of the slow component to total synaptic current was estimated to be 28%. The right trace in Fig. 3A shows NMDA EPSC in a nerveligated neuropathic mouse, together with double exponential fits to the decay phase. τ1 and τ2 were 63.5 ms and 299.8 ms, respectively. The contribution of the slow component to the total synaptic current was estimated to be 42%. The NMDA EPSCs recorded from 17 neurons in nerveligated neuropathic mice and those from 10 neurons in sham-
Fig. 3 – Decay kinetics of NMDA EPSCs in sham-operated and nerve-ligated mice. (A) Representative recordings of evoked EPSCs in a sham-operated control mouse (left panel) and a nerve-ligated neuropathic mouse (right panel). Recordings were made at a holding potential of +50 mV in the presence of bicuculline, strychnine, glycine, and CNQX. In both cases, the decay was best fitted by a double exponential function. The fitted functions are superimposed (thin line) with their time constants (slow and fast components of NMDA EPSCs). The number in parentheses represents the amplitude of the slow component relative to the total amplitude of the NMDA EPSCs. (B) Bar graphs summarizing decay kinetics of NMDA EPSCs in sham-operated control mice (shadowed bars) and nerve-ligated neuropathic mice (open bars). The left panel shows the time constant of the fast component (τ1), the middle panel shows the slow component (τ2), and the right panel shows the relative amplitude of the slow component. Bars and vertical lines indicate the mean values ± SEM. * Indicates significance at p < 0.05. ** Indicates significance at p < 0.01. NS indicates “not significant.”
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Fig. 4 – Effects of ifenprodil on NMDA EPSCs in nerve-ligated mice. (A) Representative recordings of the NMDA EPSCs evoked before (left panel) and during (right panel) the application of ifenprodil. The recordings were made at a holding potential of +50 mV in the presence of bicuculline, strychnine, glycine, and CNQX. Ifenprodil was applied by bath perfusion at a concentration of 10 μM. The decay was best fitted by a double exponential function in both cases. The fitted functions are superimposed (thin line) with their time constants (slow and fast components of NMDA EPSCs). The number in parentheses represents the relative amplitude of the slow component of the NMDA EPSCs. (B) Graphs summarizing the effects of ifenprodil on NMDA EPSCs in sham-operated control mice (filled circles) and nerve-ligated neuropathic mice (open circles). The leftmost graph shows the effect of ifenprodil on the ratio of the NMDA EPSC amplitude to the non-NMDA EPSC amplitude. The second and third graphs from the left show the effect of ifenprodil on the time constant of the fast (τ1) and slow (τ2) components of NMDA EPSCs, respectively. The rightmost graph shows its effect on the relative amplitude of the slow component of NMDA EPSCs. Symbols and vertical lines indicate the mean values ± SEM. * Indicates significance at p < 0.05. ** Indicates significance at p < 0.01. NS indicates “not significant.”
operated control mice were fitted with a double exponential function. τ1 and τ2 were 31.4 ± 6.7 ms and 201.5 ± 10.7 ms (mean ± SEM), respectively, in sham-operated control mice (Fig. 3B, leftmost and middle graphs, shadowed bars). τ1 and τ2 in nerve-ligated neuropathic mice were 68.5 ± 9.3 ms and 312.8 ± 19.5 ms, respectively (Fig. 3B, leftmost and middle graphs, open bars). τ2 in nerve-ligated neuropathic mice was statistically longer than those in sham-operated control mice (p < 0.01). The relative contribution of the slow component of NMDA EPSCs was 46.0 ± 7.2% in nerve-ligated neuropathic mice, which is significantly larger than that in sham-operated control mice (29.1 ± 8.7%, p < 0.05, Fig. 3B, graph at the extreme right).
2.5.
neurons in sham-operated control mice. Fig. 4 is an example of the agarose gel electrophoresis of the RT-PCR products obtained by single-cell RT-PCR analysis. Lanes labeled cell #1 are for a neuron from a sham-operated control mouse, and lanes labeled cell #2 are for a neuron from a nerve-ligated neuropathic mouse. The PCR product of neuron-specific enolase (NSE) mRNA was detected in all the cells examined. Of the 51 neurons in nerve-ligated mice, 15 expressed NR2A mRNA but not NR2B mRNA, 12 neurons expressed both NR2A and NR2B, and 16 neurons expressed NR2B but not NR2A; NR2A-B subunits were not detected in 8 neurons (Table 1, upper row). Of the 49 neurons in sham-operated control mice, 32 neurons expressed NR2A but not NR2B, 9 neurons expressed both NR2A and NR2B, and 2 neurons expressed NR2B but not NR2A; NR2A-B subunits were not detected in 6 neurons (Table 1, lower row). The Chi-square test indicates that the incidence of NR2A subunit expression was significantly larger in sham-operated control mice than in nerve-ligated neuropathic mice (p < 0.01). Further, the incidence of NR2B subunit expression was significantly larger in nerve-ligated neuropathic mice than in sham-operated control mice (p < 0.01).
2.6.
Effects of ifenprodil on NMDA receptor-mediated EPSC
Fig. 5A shows the effects of ifenprodil, an NR2B antagonist, at a concentration of 10 μM on an NMDA EPSC in a nerve-ligated neuropathic mouse. Ifenprodil slightly but distinctly decreased the amplitude of NMDA EPSC in this example. Fitting the double exponential function to the decay time course indicated that ifenprodil shortened the decay phase and decreased the relative contribution of the slow component to the EPSC. The effects of ifenprodil were studied in 8 neurons in nerve-ligated neuropathic mice and in 8 neurons in shamoperated control mice. Grouped data are shown in Fig. 5B, which indicates that nerve-ligated neuropathic and shamoperated control mice have significantly different susceptibilities to ifenprodil [two-way analysis of variance (ANOVA), p < 0.05]. In nerve-ligated neuropathic mice (Fig. 5B, open circles), ifenprodil decreased the amplitude of NMDA EPSC to 34.3 ± 5.1% of the control (p < 0.05); the time constant of the fast component, from 52.6 ± 7.8 ms to 39.2 ± 3.5 ms (no significant difference); the time constant of the slow component, from 302.3 ± 24.7 ms to 181.0 ± 23.2 ms (p < 0.01); and the relative contribution of the slow component to the synaptic current, from 42.9 ± 5.9% to 21.2 ± 8.1% (p < 0.05). Single-cell RT-PCR
Table 1 – Expression pattern of NMDA receptor subunits in sham-operated and nerve-ligated mice NR2A
NR2B
NR2A and NR2B
None
Total number of neurons
15
16
12
8
51
32
2
9
6
49
Single-cell RT-PCR analysis of NMDA subunits
After patch clamp recordings, we collected the neuron under investigation. Single-cell RT-PCR analysis was performed on 51 neurons in nerve-ligated neuropathic mice and on 49
Nerve-ligated mice Sham-operated mice
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Fig. 5 – Single-cell RT-PCR analysis of NMDA receptor subunit expression in sham-operated and nerve-ligated mice. Lanes labeled cell #1 are for a neuron sampled from a sham-operated control mouse. Lanes labeled cell #2 are for a neuron sampled from a nerve-ligated neuropathic mouse. The lane labeled “Marker” contains the 100-bp DNA ladder. Values in parentheses on the left indicate the expected length of the PCR products for NSE, NR2A, and NR2B.
analysis revealed that 4 of these 8 neurons expressed only NR2B mRNA, 3 of these 8 neurons expressed both NR2AB and NR2B mRNAs, and no RT-PCR product was detected in the remaining neuron. In sham-operated control mice (Fig. 5B, filled circles), ifenprodil decreased the amplitude of NMDA EPSC to 57.4% ± 11.4% of the control (p < 0.05). However, the time constants of the fast and slow components or the relative contribution of the slow component were not significantly altered by ifenprodil (p > 0.05). Single-cell RT-PCR analysis showed that 6 of these 8 neurons expressed only NR2A mRNA, 2 of these 8 neurons expressed both NR2A and NR2B mRNAs. The differential expression patterns of NMDA receptor subunits between nerve-ligated neuropathic mice and sham-operated control mice are consistent with the observed differential effects of ifenprodil.
3.
Discussion
In the present experiments, we recorded synaptically evoked NMDA EPSCs from the superficial dorsal horn neurons in the spinal cord slice. The functional properties of the NMDA
receptors expressed in the synaptic regions of nerve-ligated neuropathic mice were compared with those of shamoperated control mice.
3.1. The functional properties of NMDA EPSCs changed in nerve-injured mice The glutamatergic EPSCs evoked by the electrical stimulation of adjacent fibers were recorded from neurons located in the superficial dorsal horn. The glutamatergic EPSCs were composed of two components, NMDA EPSC and nonNMDA EPSC. The ratio of the amplitude of the NMDA EPSC to that of the non-NMDA EPSC was significantly larger in nerveligated neuropathic mice than in sham-operated control mice. Although the fast and slow time constants of NMDA EPSCs recorded in sham-operated control mice varied considerably among neurons, they are in the range of the previously reported values in rat dorsal horn neurons which express NR2A subunit-containing NMDA receptors (Bardoni et al., 1998; Momiyama, 2000). The results also showed that the NMDA EPSCs had slower decay time courses in nerve-ligated neuropathic mice than in sham-operated control mice.
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A detailed analysis of the peripheral nerve injury-induced alterations of the properties of the NMDA receptors in the spinal cord dorsal horn was previously reported (Guo and Huang, 2001; Isaev et al., 2000; Karlsson et al., 2002). For example, Guo and Huang made patch clamp recordings from isolated spinal dorsal horn neurons and analyzed whole cell currents that were induced by pressure-applied NMDA. Peripheral inflammation induced by injections of complete Freund's adjuvant reduced the magnesium block of NMDA responses and shifted the voltage dependence of the NMDA receptors toward the hyperpolarized direction (Guo and Huang, 2001). Karlsson et al. recorded NMDA receptor-mediated currents evoked by glutamate application in single neurons dissociated from the rat spinal cord. They reported that despite minor changes in the dose–response relation of glutamate-evoked NMDA receptor-mediated currents after the nerve lesion, there were no significant differences in the density and magnesium block of the current between the sham-operated control and nerve-ligated neuropathic rats (Karlsson et al., 2002). Isaev et al. recorded whole cell membrane currents and calcium transient in superficial dorsal horn neurons in the rat spinal cord slice preparation. They found that spinal nerve ligation produces prolonged facilitation of membrane currents and calcium transient induced by bath application of NMDA. They suggested that the increase in calcium signaling mediated by NMDA receptors is involved in the development of neuropathic pain following spinal nerve injury (Isaev et al., 2000). The present results are in line with this previous report and provide direct evidence that the relative contribution of the NMDA receptor to excitatory synaptic transmission in the superficial dorsal horn increases with peripheral nerve injury.
3.2. Effects of the NR2B subunit-specific antagonist ifenprodil on NMDA EPSCs in nerve-ligated neuropathic mice The NR2B subunit-specific NMDA receptor antagonist ifenprodil (10 μM) reduced the amplitude of the NMDA EPSCs and shortened their decay phase. These effects of ifenprodil were more prominent in nerve-ligated neuropathic mice than in sham-operated control mice. Momiyama investigated the neurons in the superficial dorsal horn of adult rats and reported that ifenprodil (10 μM) reduced whole cell current induced by NMDA application, but ifenprodil did not affect synaptically evoked NMDA EPSCs. In contrast to this, we observed that ifenprodil (10 μM) reduced the amplitude of the NMDA EPSCs. The contrasting results of ifenprodil's effects on synaptically evoked NMDA EPSCs in the spinal cord dorsal horn, although not fully clear, may be partially explained by the differences in stimulation methods. In this study, NMDA EPSCs were evoked by a glass pipette placed near the recorded neurons, while Momiyama stimulated a dorsal root by a suction electrode. Dorsal root stimulation and intraspinal stimulation may activate different groups of synapses with different subunit composition, or they may activate the NMDA receptors to different extents. It has been suggested that the effects of ifenprodil depend on the extent of NMDA receptor activation (Kew et al., 1996; Zhang et al., 2000). At low NMDA concentration, ifenprodil enhances
NMDA-induced currents, while at high NMDA concentration, the NMDA-induced currents are inhibited. Nagy et al. (2004) analyzed the synaptic distribution of the NR2B subunit in the rat lumbar spinal cord by using an antigen-unmasking technique. They demonstrated that the NR2B subunit is concentrated in the superficial dorsal horn, and suggested that there are high levels of NR2B in many synaptic regions. Our observation that ifenprodil suppressed synaptically evoked NMDA EPSCs seems consistent with this report.
3.3. Single-cell RT-PCR analysis of NMDA receptor subunits expressed in the superficial dorsal horn The previously reported single-cell RT-PCR analysis on neurons dissociated from the spinal cord dorsal horn showed that fewer neurons express the NR2A in peripheral nerveinjured rats as compared to those in the control rats, and the incidence of neurons expressing the NR2B increased proportionally in nerve-injured rats (Karlsson et al., 2002). It has also been reported that, in mice, the expression level of NR2A in the dorsal horn decreased after injection of formalin into the hindpaw (Gaunitz et al., 2002). Furthermore, exaggerated nociceptive states induced by peripheral nerve injury or tissue inflammation is known to be mediated by the activation of NR2B subunits distributed in the spinal cord dorsal horn (Boyce et al., 1999; Chizh et al., 2001; Sakurada et al., 1998; Tan et al., 2005; Taniguchi et al., 1997). These previous reports prompted us to compare the expression pattern of NR2A and NR2B in the superficial dorsal horn neurons between shamoperated control mice and nerve-ligated neuropathic mice. The results showed that the ratio of the number of neurons expressing NR2A subunit decreased, whereas the ratio of the number of neurons expressing NR2B subunit increased after nerve ligation. This observation is consistent with the abovementioned report of single-cell RT-PCR analysis on dissociated spinal cord neurons of neuropathic rats.
3.4. Possible mechanisms for the observed alterations in the functional properties of NMDA receptors in nerve-ligated neuropathic pain Substantial evidence indicates that the neuropathic pain induced by peripheral nerve injury is accompanied by alterations in synaptic transmission in the spinal cord dorsal horn. The alterations associated with neuropathic pain include phosphorylation of NMDA receptors (Guo et al., 2002; Zou et al., 2002), altered NMDA receptor subunit expression pattern (Gaunitz et al., 2002; Karlsson et al., 2002), conversion of silent synapses containing only NMDA receptors to functional synapses (Baba et al., 2000), and an increase in NMDAmediated current (Isaev et al., 2000). Although the precise mechanisms for the presently observed alterations in the functional properties of NMDA EPSCs in nerve-ligated neuropathic mice are not clear, our eletrophysiological observations, together with single-cell RT-PCR analysis appear to indicate that the subunit composition of the NMDA receptors expressed in the synaptic region might change after nerve injury. The results of single-cell RT-PCR analysis show that the incidence of NR2A decreased and that of NR2B increased after
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peripheral nerve injury leading to a slower decay time course and a higher susceptibility to ifenprodil, as observed in the nerve-ligated neuropathic mice. The NR2 subunits are known to determine the kinetic properties of the NMDA receptors (Cathala et al., 2000; Misra et al., 2000; Vicini et al., 1998; Wyllie et al., 1998). Increased NR2A subunit expression and decreased NR2B subunit expression accelerate NMDA EPSCs (Cathala et al., 2000; Cull-Candy and Leszkiewicz, 2004; Flint et al., 1997). Developmental changes in the subunit composition of the NMDA receptors have been reported. During postnatal development, insertion of the NR2A subunit and loss of the NR2B subunit shorten the decay time course of the NMDA EPSCs (Cathala et al., 2000). Furthermore, even after neuronal development, the NMDA receptor subunit expression pattern was reported to change in an activity-dependent manner. For example, it has been shown that suppressing neuronal activity results in the re-expression of NR2B subunits (Ehlers, 2003; Kiyosue et al., 2004). It is conceivable that alterations in synaptic activity resulting from peripheral nerve ligation might alter the NMDA receptor subunit expression pattern in the spinal cord dorsal horn. Finally, NR1 and NR2A subunits are also reported to be involved in neuropathic pain. Further studies on these NMDA receptor subunits would provide clues for further understanding the cellular mechanisms of neuropathic pain induced by peripheral nerve injury.
In some mice, ifenprodil (2000 pmol, Tan-No et al., 2000), a selective antagonist against the NMDA receptor 2B subunit, was administered intrathecally between lumbar segment L5 and L6 according to the procedure described by Hylden and Wilcox (1980) under unanesthetized conditions. The intrathecal location of the needle tip was affirmed by a characteristic flick of the tail. The withdrawal thresholds were measured 10– 20 min after the intrathecal administration of ifenprodil. Ifenprodil (RBI, MA, U.S.A.) was dissolved in 1% dimethyl sulphoxide (DMSO) in saline. Some nerve-ligated mice were injected with the vehicle only. The intrathecal administration and threshold measurements were performed 7 (n = 12), 8 (n = 12), 9 (n = 10), and 10 (n = 10) days after the partial nerve ligation, and all the data were pooled for further analysis.
4.4.
Experimental procedures
4.1.
Animals
Experiments were conducted using adult male ICR mice (7–8 weeks old) that were kept at controlled room temperature under a 12-h light/dark cycle. The care and use of the animals were in accordance with institutional guidelines and the guidelines of the International Association for the Study of Pain (Zimmermann, 1983).
4.2.
Surgery
The mice were anesthetized by an intraperitoneal injection of sodium pentobarbital (50 mg/kg). Partial ligation of the left sciatic nerve was made according to the protocol for rats described by Seltzer et al. (1990). In sham-operated control mice, the sciatic nerve was exposed but not ligated.
4.3.
Assessment of behavioral mechanical
Mice were placed on an elevated plastic mesh floor covered with a clear plastic box (height, 15 cm; diameter, 12 cm). The withdrawal threshold to mechanical stimulation was determined. The mechanical stimulus was applied from underneath to the plantar aspect of the hindlimb, ipsilateral to the partial nerve ligation by means of an Elctro von Frey (Model 1601; IITC Inc., Woodland Hills, CA). The lowest force from five tests that induced a withdrawal response was considered the withdrawal threshold. The measurements were made from 2 days before the surgical operation through 27 days after the surgical operation.
Preparation of spinal cord slices
Transverse slices were obtained 7–10 days after sciatic nerve ligation. Segments of the lumbosacral (L4-S1) spinal cord were removed under deep ether anesthesia. A Vibratome (Dosaka EM, Japan) was used to cut transverse slices (350–450 μm) in Krebs solution at 4 °C. The Krebs solution was equilibrated with 95% O2 and 5% CO2 and contained the following (in mM): NaCl, 113; KCl, 3; NaHCO3, 25; NaH2PO4, 1; CaCl2, 2; MgCl2, 1; Dglucose, 11; pH 7.4.
4.5.
4.
99
Whole cell recordings
After a 1-h incubation period in Krebs solution at 37 °C, the slices were mounted onto a recording chamber on a microscope stage and continuously perfused with Krebs solution. Conventional tight-seal whole cell recordings were made from visually identified neurons located in the superficial dorsal horn (laminae II and III) of the spinal cord slice using infrared DIC videomicroscopy as previously described (Wake et al., 2001). Since no apparent difference in the characteristics of the NMDA receptor-mediated postsynaptic currents was observed, data obtained from the neurons in laminae II and III were pooled for analysis. Patch pipettes were made from thin-wall borosilicate glass capillaries pulled on a Flaming Brown pipette puller (Sutter Instruments, Novato, CA, U.S.A.) and filled with a solution of the following composition (in mM): K gluconate, 123; KCl, 14; Na gluconate, 2; EGTA, 1; HEPES, 10; pH neutralized to 7.4 with KOH. The DC resistance of the pipettes filled with the internal solution was 5–10 MΩ. The membrane potential value was corrected for junction potential. Fast and slow capacitances were neutralized. The series resistance was compensated by 60%. The access resistance (11–20 MΩ) was continuously monitored and data were discarded when the value changed by more than 10%. EPSCs were evoked at 0.1 Hz by using a stimulating electrode filled with 1 M NaCl with its tip (diameter, ca. 3 μm) placed at the dorsolateral margin of the spinal cord, 100– 200 μm away from the recording site. All recordings were made in the presence of strychnine (Sigma, 0.5–1.0 μM), bicuculline (Sigma, 20 μM), and glycine (Sigma, 5 μM). To record the non-NMDA receptor-mediated component of EPSCs (non-NMDA EPSCs), each neuron was held at −70 mV in the presence of AP5 (50 μM). To record the NMDA receptor-
100
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mediated component of EPSCs (NMDA EPSCs), each neuron was held at +60 mV in the presence of CNQX (10 μM). EPSCs were recorded in the voltage clamp mode using an Axopatch 200B patch clamp amplifier (Axon Instruments). Data were sampled at a rate of 10.0 kHz through a Digidata 1230 interface (Axon Instruments). pCLAMP (Axon Instruments) was used to analyze the data.
4.6.
Single-cell RT-PCR
After whole cell recordings were made, the neurons were aspirated into another pipette following a previously described protocol (Tsuchiya et al., 1999). The collecting pipette had a tip diameter of about 3–5 μm and contained 2 μl of Ca2+- and Mg2+-free phosphate-buffered saline solution. The neurons were then ejected into thin-walled autoclaved PCR tubes by applying a gentle positive pressure, and immediately frozen and stored at − 80 °C until use. The PCR tubes contained 2 μl MgCl2 (25 mM), 2 μl 10 PCR buffer (200 mM Tris–HCl, 500 mM KCl), 0.5 μl RNase inhibitor (40,000 units/ml), 2 μl nonionic detergent IGEPAL CA-630 (5%), and 5 μl DEPCtreated water. On the following day, lysis was performed using IGEPAL CA-630 at room temperature for 5 min and the reversetranscription mixture was added. It contained 1 μl oligo (dT) primers (0.5 μg/μl), 2 μl mixed deoxynucleotide triphosphates (dNTPs, 10 mM), 2 μl dithiothreitol (0.1 M), 0.5 μl RNase inhibitor (40,000 units/ml), and 1 μl SuperScript II RT (200 Units/μl). The reaction mixture was incubated at 42 °C for 50 min. Subsequently, the sample was heat inactivated at 70 °C for 15 min. The PCR reactions were performed after the protocol of Fukushima et al. (2005) in a 50-μl volume containing 20 mM Tris–HCl, 50 mM KCl, 2.5 mM MgCl2, 0.2 mM dNTPs, and 2.5 units Taq DNA polymerase. The entire RT product was used for the first PCR, and 1 to 2 μl of the first PCR product was used for the second PCR. The primers used targeted three genes, NSE, and the NMDA receptor subunits NR2A and NR2B. NSE was used as a positive control. Primers used for detection of NSE were 5′-ATAGTGGGCGATGACC TGAC-3′ and 5′-ATGAACGTGTCCTCCGTTTC-3′ (200-bp product; GenBank accession # NM013509). Primers used for NR2A subunit detection were 5′-GCTACGGGCA GACAGAGAAG-3′ and 5′-GTGGTTGTCATCTGGCTCAC-3′ (257-bp product; GenBank accession # NM008170). Primers used for NR2B subunit detection were 5′-GCTACAACACC CACGAGAAGAG-3′ and 5′-GAGAGGGTCCACGCTTTCC-3′ (314-bp product; GenBank accession # NM008171). These primers were used for both the first and second PCR amplifications. Each primer was individually used in the second PCR. The concentration of primers was 20 nM each in the first PCR and 200 nM in the second PCR. The thermal cycler (Gene Amp 2400, Perkin Elmer) was programmed for 35–40 cycles of 1 min denaturation (94 °C), 1 min annealing (54–59 °C), and 1 min elongation (72 °C). The second PCR products were visualized by staining with ethidium bromide and separated by agarose gel electrophoresis. All the products were sequenced using dye terminator chemistry (Applied Biosystems) and a DNA sequencer (Model 377, ABI) and matched the published sequences.
All the reagents for the RT-PCR, except the RNase inhibitor (TOYOBO), IGEPAL CA-630 (Sigma), and Taq DNA polymerase (Takara), were obtained from GIBCO BRL.
4.7.
Statistical analysis
Results are expressed as mean ± SEM. Statistical analysis of the results was performed by one-way or two-way analysis of variance (ANOVA) followed by post hoc multiple comparison (Tukey test). Student's t-test and kai2 test were also used when appropriate. Differences were considered significant at the level of p < 0.05.
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