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Changes in VGLUT2 expression and function in pain-related supraspinal regions correlate with the pathogenesis of neuropathic pain in a mouse spared nerve injury model
brain research 1624 (2015) 515–524
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Changes in VGLUT2 expression and function in pain-related supraspinal regions correlate with the pathogenesis of neuropathic pain in a mouse spared nerve injury model Zhi-Tong Wang, Gang Yun, Hong-Sheng Wang, Shou-Pu Yi, Rui-Bin Sun, Ze-Hui Gong State Key Laboratory of Toxicology and Medical Countermeasures, Beijing Institute of Pharmacology and Toxicology, 27 Taiping Road, Beijing 100850, People’s Republic of China
art i cle i nfo
ab st rac t
Article history:
Vesicular glutamate transporters (VGLUTs) control the storage and release of glutamate, which
Accepted 13 August 2015
plays a critical role in pain processing. The VGLUT2 isoform has been found to be densely
Available online 20 August 2015
distributed in the nociceptive pathways in supraspinal regions, and VGLUT2-deficient mice exhibit an attenuation of neuropathic pain; these results suggest a possible involvement of
Keywords:
VGLUT2 in neuropathic pain. To further examine this, we investigated the temporal changes in
Neuropathic pain
VGLUT2 expression in different brain regions as well as changes in glutamate release from
VGLUT2
thalamic synaptosomes in spared nerve injury (SNI) mice. We also investigated the effects of a
Supraspinal brain regions
VGLUT inhibitor, Chicago Sky Blue 6B (CSB6B), on pain behavior, c-Fos expression, and
Thalamus
depolarization-evoked glutamate release in SNI mice. Our results showed a significant elevation
Glutamate release
of VGLUT2 expression up to postoperative day 1 in the thalamus, periaqueductal gray, and
Chicago Sky Blue 6B
amygdala, followed by a return to control levels. Consistent with the changes in VGLUT2 expression, SNI enhanced depolarization-induced glutamate release from thalamic synaptosomes, while CSB6B treatment produced a concentration-dependent inhibition of glutamate release. Moreover, intracerebroventricular administration of CSB6B, at a dose that did not affect motor function, attenuated mechanical allodynia and c-Fos up-regulation in pain-related brain areas during the early stages of neuropathic pain development. These results demonstrate that changes in the expression of supraspinal VGLUT2 may be a new mechanism relevant to the induction of neuropathic pain after nerve injury that acts through an aggravation of glutamate imbalance. & 2015 Elsevier B.V. All rights reserved.
Abbreviations: VGLUT,
vesicular glutamate transporter; SNI,
amino acid transporter; PAG, PB,
phosphate-buffer; PBS,
periaqueductal gray; mPFC,
spared nerve injury; CSB6B,
medial prefrontal cortex; ACSF,
phosphate-buffered saline; 4-AP,
excitatory
4-aminopyridine; L-trans-2,4-PDC, L-trans-2,4-Pyrrolidine dicarboxylic
acid n Corresponding authors. Fax: þ86 10 68211656. E-mail addresses: [email protected] (G. Yu), [email protected] (R.-B. Su). http://dx.doi.org/10.1016/j.brainres.2015.08.010 0006-8993/& 2015 Elsevier B.V. All rights reserved.
Chicago Sky Blue 6B; EAAT, artificial cerebrospinal fluid;
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1.
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Introduction
Glutamatergic transmission and homeostasis are regulated by a glutamate transporter system that includes excitatory amino acid transporters (EAATs) and vesicular glutamate transporters (VGLUTs). EAATs remove glutamate from the neuronal synapse into glia and neurons to terminate excitatory signaling, whereas VGLUTs transport glutamate into vesicles prior to exocytotic release. Three distinct isoforms of VGLUTs have been identified (Ni et al., 1994; Takamori et al., 2001; Schafer et al., 2002), with VGLUT1 and VGLUT2 accounting for most of the presumed excitatory glutamatergic terminals in the central nervous system, whereas VGLUT3 is diffusely distributed in the brain, defining a discrete subpopulation of non-glutamatergic neurons (Kaneko and Fujiyama, 2002; Schafer et al., 2002). In adult rodents, VGLUT2 is most abundant in the diencephalon, brainstem, and spinal cord (Sakata-Haga et al., 2001; Kaneko and Fujiyama, 2002; Landry et al., 2004). The characteristic distribution pattern of VGLUT2 seems to be generally coincident with the nociceptive pathways, suggesting that VGLUT2 is involved in the signaling of pain. In addition, several studies in heterozygous mice have demonstrated that VGLUT2 deficiency results in attenuation or deletion of some neuropathic pain symptoms, unlike the effects of a VGLUT1 signaling impairment (Moechars et al., 2006; Leo et al., 2009). However, these results from genetically modified mice may be confounded by multiple factors, such as adaptation or compensation during development. Moreover, no study has investigated in wild-type animals possible time-dependent changes in VGLUTs in the central nervous system during the pathophysiological process of neuropathic pain development. The supraspinal regions are especially understudied in this connection. In the transmission of pain, nociceptive information from the spinal cord terminates in the thalamus, which is a key relay station for transmission to the cerebral cortex and periaqueductal gray (PAG), which are implicated in descending pain modulation (Millan, 2002). As to affect and cognition in pain processes, the amygdala modulates cortical functions associated with pain-induced mood disorders (Yalcin et al., 2014), while the medial prefrontal cortex (mPFC) is crucial for pain-related perception (Metz et al., 2009). Importantly, the thalamus, in which 90% of the excitatory synaptic response depends on VGLUT2, has been demonstrated to exhibit immediate reorganization after partial nerve ligation (Brüggemann et al., 2001) and abnormal discharge patterns in neuropathic pain models (Guilbaud et al., 1990). In addition, it has been reported that reduction or loss of VGLUT2 expression leads to reduction in the quantal size of glutamate release in thalamic neurons and is associated with attenuation of neuropathic pain in vivo (Moechars et al., 2006). In this study, we investigated temporal changes in VGLUT2 expression in pain-related brain areas in SNI mice by immunohistochemistry. In addition, Chicago Sky Blue 6B (CSB6B) was used as a pharmacological tool to examine whether inhibition of VGLUT activity would result in attenuation of thalamic glutamate release and pain behaviors. These results provide further evidence that VGLUT2 is associated with neuropathic pain.
2.
Results
2.1.
SNI-induced mechanical allodynia
The 25 mice that received SNI surgery were divided into 5 subgroups: SNI-0.5 d, SNI-1.0 d, SNI-1.5 d, SNI-3.0 d, and SNI7.0 d. We then validated that all the mice developed mechanical hypersensitivity to von Frey stimulation and no significant difference existed among these subgroups at the corresponding time points. Thus, only behavioral data in the SNI-7.0 d group are shown (Fig. 1). Repeated-measures two-way ANOVA showed significant effects of treatment (F [1,32]¼ 1025; po0.001) and a Bonferroni post hoc test revealed a significant decrease in the mechanical withdrawal threshold of the hind paws ipsilateral to the SNI surgery sides on postoperative days 0.5 (po0.001), 1.0 (po0.001), 1.5 (po0.001), 3.0 (po0.001), and 7.0 (po0.001).
2.2. Changes in VGLUT2 expression in pain-related brain areas after SNI The specificity of the VGLUT2 antibody has been validated by the pre-adsorption study (Supplementary Fig. 1). In the analyzed supraspinal areas, VGLUT2 immunostaining is punctuate, compatible with nerve terminals in the vicinity of neuronal cell bodies. Fig. 2 shows the changes in VGLUT2 expression at different times after SNI. Two-way ANOVA shows a significant effect of treatment in thalamus (F [1,40]¼ 20.81, po0.001) and PAG (F[1,40]¼ 4.67, po0.05). The Bonferroni post hoc test shows significant differences between sham and SNI mice in thalamus (po0.001) and PAG (po0.01) on postoperative day 1. Although two-way ANOVA does not show a significant effect of treatment in ipsilateral or contralateral amygdala, planned post hoc comparisons reveal a significant difference between sham and SNI mice on postoperative day 1 (contra, po0.01; ipsi,
Fig. 1 – Spared nerve injury (SNI) reduces paw withdrawal threshold. This was demonstrated by stimulating the sural nerve territory with a series of von Frey filaments. The mechanical threshold of SNI mice was significantly lower than that of the sham mice on postoperative days 0.5, 1.0, 1.5, 3.0, and 7.0, expressed as means7SEM. Note that the spacing of the time points is not to scale in this and subsequent figures n¼ 5; ***po0.001 compared with the sham control.
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po0.05). The up-regulation of VGLUT2 expression is transient and the level returns to baseline by 36 h (p40.05), remaining there for the rest of the experiment. In mPFC, there are no significant alterations in VGLUT2 staining, although a slight increase is observed on day 1.
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2.3. Effects of CSB6B on neuropathic pain and motor function in mice Fig. 3 shows the effects of CSB6B on SNI-induced neuropathic pain following different treatment schedules. Intracerebro-
Fig. 2 – Changes in VGLUT2 expression in supraspinal areas following spared nerve injury. The left panels show representative photomicrographs taken from SNI and sham mice (scale bar, 100 μm), showing an increase in VGLUT2 in SNI mice on postoperative day 1 (SNI-D1) within the analyzed brain regions, including thalamus (Thal), periaqueductal gray (PAG), amygdala (Amy), and medial prefrontal cortex (mPFC) compared with the sham control (Sham), followed by a return to sham control levels when examined on postoperative day 7 (SNI-D7). Plots on the right represent the quantitative labeling analysis expressed as relative mean density (n ¼5 for each group). Data are calculated as fold changes in mean density, and expressed as means7SEM; *po0.05; **po0.01; ***po0.001 compared with the sham control.
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ventricular administration of CSB6B (5 μg) at 12- h intervals starting from day 7 after SNI has no effect on established mechanical allodynia (Fig. 3B). The mice treated with CSB6B starting at the time of SNI, however, show a significant difference in the development of mechanical allodynia (F [1,84]¼ 8.17; po0.05). Bonferroni post hoc tests indicate that the pain threshold is significantly higher in CSB6B-treated mice than in their ACSF-treated counterparts on postoperative day 1.0 (0.6770.09 vs. 0.1470.04, po0.001). Nevertheless, considerable mechanical allodynia develops in CSB6B-treated mice, and no significant difference exists between the two groups from postoperative day 1.5–3.5 (p40.05, Fig. 3C). To exclude possible direct effects of CSB6B on motor function, we compared the rotarod performances of mice treated with ACSF and mice treated with CSB6B. No significant difference is seen between these two groups (F[1,56]¼ 0.09; p40.05; Fig. 3D), indicating that the apparent antinociceptive effects of CSB6B are not due to effects on motor function.
2.4. Effects of CSB6B on SNI-induced c-Fos expression in pain-related brain areas To further increase our confidence that CSB6B affects the degree of hyperalgesia after SNI surgery, the expression of
c-Fos in pain-related brain areas was tested by immunohistochemistry. Two-way ANOVA indicates significant effects of treatment in thalamus (F[2,36]¼9.70, po0.001), PAG (F[2,36]¼ 16.32, po0.001), contralateral amygdala (F[2,36]¼ 5.13, po0.05), ipsilateral amygdala (F[2,36]¼ 3.67, po0.05), contralateral mPFC (F[2,36]¼3.94, po0.05), and ipsilateral mPFC (F [2,36]¼ 6.57, po0.01) (Fig. 4). The Bonferroni post hoc test shows an SNI-induced up-regulation of c-Fos expression in thalamus (po0.05) and PAG (po0.001) on postoperative day 1, and in PAG (po0.01) and contralateral mPFC (po0.01) on postoperative day 1.5. Intracerebroventricular administration of CSB6B produces a tendency toward reduction in c-Fos expression in all areas analyzed and statistical significance is reached in thalamus on postoperative day 0.5 (po0.05) and in ipsilateral mPFC on postoperative day 1.5 (po0.05).
2.5. Effects of CSB6B on glutamate release in thalamic synaptosomes from SNI mice The depolarizing agent 4-AP releases neurotransmitter from the synaptic terminal in the presence of Ca2þ by inducing repetitive spontaneous Naþ-channel-dependent depolarizations, resembling the in vivo process of depolarization (Nicholls, 1998). Because EAAT expression was reported to change during SNI development, we added L-trans-2,4-PDC,
Fig. 3 – Effects of intracerebroventricular CSB6B on pain behavior and motor function. A: The distribution of CSB6B in the ventricular system after intracerebroventricular administration. After behavioral tests, mice were decapitated and the brains were removed to verify the placement of the cannula. Only the data from those animals with dispersion of the dye throughout the ventricles were used. B: Effects of CSB6B (5 μg in 5 μL ACSF, twice daily starting from postoperative day 7) on mechanical allodynia (n ¼5). C: Effects of CSB6B (5 μg, twice daily from postoperative day 1 to day 3.5 following an initial treatment immediately after the SNI) on mechanical allodynia (n ¼ 5 for control and n¼ 11 for CSB6B treatment). Data are expressed as means7SEM; ***po0.001 compared with the sham control. D: Effects of CSB6B on motor function. Mice injected i.c.v. with CSB6B (5 μg, twice daily) or ACSF show no differences in their ability to remain on an accelerating rotarod (n¼ 10). Data are expressed as means7SEM.
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Fig. 4 – Changes in numbers of c-Fos-immunoreactive cells in brain regions of SNI mice after CSB6B administration. The first three columns of panels show sections of thalamus and other pain processing areas from sham control and spared-nerve injured animals treated for 0.5 days with i.c.v. injections of ACSF or 5 μg of CSB6B (scale bar, 200 μm). Plots on the right represent the quantitative labeling analysis expressed as c-Fos-positive neurons/section (n ¼5 for each group). Data are expressed as mean7SEM; *po0.05 compared with the sham control; ♯po0.05 compared with the ACSF-treated group. which inhibits all five EAAT subtypes, to exclude the influence of plasma membrane reuptake on the concentration of glutamate in the incubation solution (Bridges et al., 1999). Fig. 5 shows the effects of SNI (on postoperative day 1) and CSB6B on 4-AP-evoked glutamate release in thalamic synaptosomes. One-way ANOVA shows a significant effect of treatment (F[3,16]¼ 70.15, po0.001). The Bonferroni post hoc test reveals a significant increase in glutamate release in SNI mice (po0.05) relative to sham-operated mice. Preincubation with CSB6B (20 μM) significantly reduces glutamate release in sham (po0.001) and SNI mice (po0.01, Fig. 5A). Fig. 5B shows
that the inhibitory effect of CSB6B is concentration-dependent, with an IC50 value of 25.7 μM.
3.
Discussion
To demonstrate the relation between VGLUT2 and neuropathic pain, we examined changes in VGLUT2 expression in pain-related brain areas after SNI surgery. The results showed an increase in VGLUT2 expression during an early postoperative stage followed by a return to control levels.
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Fig. 5 – CSB6B inhibits 4-AP-induced glutamate release from nerve terminals in mouse thalamus. A: Quantitative comparison of 4-AP-evoked glutamate release from thalamic synaptosomes 1 day after sham and SNI surgery. The presence of 20 μM CSB6B inhibits 4-AP-induced glutamate release in sham and SNI mice. Results are mean7SEM of 5 independent experiments; * po0.05; ***po0.001 compared with the sham control; ♯♯po0.01 compared with the SNI group. B: The concentration-response curve of the inhibitory action of CSB6B on 4-AP-evoked glutamate release. Results are mean7SEM of 5 independent experiments and the curve was fitted by nonlinear regression analysis with Prism 5.0 (GraphPad Software, Inc., San Diego, CA).
Correspondingly, inhibiting the activity of VGLUT2 reduced glutamate release from thalamic synaptosomes and attenuated the initial mechanical allodynia. Up-regulation of glutamatergic signaling has been proposed as a crucial mechanism of neuropathic pain. It has been demonstrated that nerve injury results in changes in the expression, distribution, and function of ionotropic and metabotropic glutamate receptors (Yogeeswari et al., 2009), as well as in the expression and uptake activity of EAATs (Sung et al., 2003), effects jointly contributing to changes in regional glutamate neurotransmission and pain behavior. More recently, a role for VGLUT2 in neuropathic pain has been suggested by studies of genetically modified mice. The heterozygous mice, which had a partial VGLUT2 deficiency, showed unchanged motor function, learning, memory, acute nociception, and inflammatory pain. However, they showed an absence of mechanical and cold allodynia after spared nerve injury (Moechars et al., 2006; Leo et al., 2009). In addition, conditioned knockout of VGLUT2 in DRG neurons attenuates physiological and pathological pain in mice (Liu et al., 2010; Scherrer et al., 2010). The present study further investigates the role of VGLUT2 in neuropathic pain, focusing on the expressional and functional changes in VGLUT2 in selected brain areas of wild-type SNI mice. The distribution of VGLUT2 supports the hypothesis that it plays a role in the processing of nociceptive signals. In the dorsal root ganglion, VGLUT2 is expressed by the majority of nociceptors (Scherrer et al., 2010). In the spinal cord, VGLUT2 is widely expressed, with the highest density in laminae I–II (Persson et al., 2006). In the brain, VGLUT2 mRNA and protein expression was reported to predominate in the thalamus, to be widely distributed in several PAG regions, and to be present in certain amygdaloid nuclei and cortical layers (Kaneko and Fujiyama, 2002; Wallen-Mackenzie et al., 2009;
Oka et al., 2012). In addition, a study in mutant mice shows that VGLUT2-mediated neurotransmission also participates in the neuronal circuitry of higher brain functions, despite its limited expression in forebrain areas (Wallen-Mackenzie et al., 2009). Consistent with previous studies, we observed significant VGLUT2 staining in the above pain-related areas and VGLUT2 seems to be expressed mainly in afferent nerves arising from lower central nervous system (Graziano et al., 2008). Moreover, we found that VGLUT2 expression showed an increase in thalamus, PAG, and amygdala, with a maximum extent of up-regulation observed in thalamus on postoperative day 1, suggesting a molecular rearrangement of excitatory transmission in these brain areas after peripheral nerve injury (Marcello et al., 2013). Although the underlying mechanisms remains to be determined, we speculate that the up-regulation of VGLUT2 may be related to changes in brain-derived neurotrophic factor (BDNF), because BDNF has been reported to increase VGLUT expression in cultured neurons (Melo et al., 2013) and to be upregulated in the central nervous system starting from day 1 after SNI (Zhou et al., 2014). We also found that the up-regulation of VGLUT2 lasted for a very short time, and was followed by a return to control levels, which may be due to inactivation of the TrkB signaling pathway (Melo et al., 2013). This may be a compensatory effect preventing over-activation of glutamatergic neurotransmission. It has been demonstrated that the expression of EAATs is altered after nerve injury in the spinal cord and some brain structures, and contributes to a disturbance of glutamate clearance and to neuropathic pain (Sung et al., 2003; Marcello et al., 2013). However, the changes in VGLUT2 expression in neuropathic pain and their relationship to glutamate release are not thoroughly understood. Considering its crucial role in pain sensation, the thalamus was selected to demonstrate
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the functional consequence of VGLUT2 up-regulation. We observed an increase in depolarization-induced glutamate release from synaptosomes prepared from thalamic tissue one day after SNI. This finding was in agreement with a study in genetically modified mice demonstrating that a deficiency of VGLUT2 protein resulted in abolition of neuropathic pain and a reduction in miniature EPSC amplitudes, indicating that VGLUT protein levels determine the glutamate content loaded into single vesicles and the size of the subsequent quantal release (Moechars et al., 2006). Our findings further confirm the role of VGLUT2 in neuropathic pain, by demonstrating that the SNI-induced increase in VGLUT2 expression led to an increase of glutamate release in the thalamus and thus contributed to the development of pain behavior. CSB6B is an azo dye that potently and selectively inhibits the function of VGLUTs without affecting plasma membrane transporters (Thompson et al., 2005; Roseth et al., 1995). Previously, we observed that i.c.v. administration of CSB6B did not affect the acute phasic pain but attenuated tonic inflammatory pain in mouse models (Yu et al., 2013). In this study, behavioral assays further showed that inhibiting the function of VGLUTs significantly reduced the development of mechanical allodynia in the initial period of neuropathic pain. The inferred antinociceptive effects of CSB6B were corroborated by changes in expression of c-Fos, a painrelated biochemical marker (Bullitt, 1990; Munglani et al., 1999), and by the absence of a direct influence on motor function. The inhibitory effects of CSB6B on thalamic glutamate release that we observed provide a plausible mechanism for the behavioral results. Although the inhibitory effect of CSB6B is not specific to one VGLUT isoform, we speculate that it exerted its effects by inhibiting the enhancement of glutamate neurotransmission caused by VGLUT2 up-regulation, as VGLUT2 is mainly expressed in pain-related brain areas and in particular 90% of thalamic excitatory synaptic response depends on VGLUT2. Our findings are in agreement with results from genetically engineered mice (Moechars et al., 2006; Leo et al., 2009). The effects of CSB6B seem to be related to upregulated VGLUT2 expression during the early stage of neuropathic pain, and the subsequent decay of its effects are consistent with the subsequent expression decreases in VGLUT2. The lack of effects of CSB6B along with normalized VGLUT2 expression during the later stages of neuropathic pain suggest that VGLUT2 mainly participates in the initiation of neuropathic pain, in advance of a variety of other factors dynamically changing during the pain development process (Latremoliere and Woolf, 2009).
4.
Conclusions
We have demonstrated that SNI can induce time-dependent changes in VGLUT2 expression in pain-related brain areas and that up-regulation of VGLUT2 increases presynaptic glutamate release in the thalamus. CSB6B produced antinociceptive effects on neuropathic pain in its initial stages, simultaneously decreasing depolarization-evoked glutamate release from thalamic synaptosomes. Therefore, the findings suggest a possible correlativity between VGLUT2 and neuropathic pain in supraspinal regions and a possibility of treating
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neuropathic pain by inhibiting VGLUTs to reduce glutamate availability.
5.
Experimental procedures
5.1.
Animals
All experiments were approved by the local ethical committee and the Institutional Review Committee on Animal Care, and were conducted in accordance with their guidelines for the use of experimental animals. Eight-week-old C57BL/6 male mice (Beijing Animal Center, Beijing, China) were housed at constant temperature (2172 1C) on a 12-h light/ dark cycle with free access to food and water.
5.2.
Spared nerve injury (SNI) surgery
Under pentobarbital sodium (40 mg/kg, i.p.) anesthesia, mice underwent the following surgery according to the method of Decosterd and Woolf (2000). The three terminal branches of the sciatic nerve (the common peroneal, tibial, and sural nerves) were exposed after skin and muscle incision. Both the common peroneal and tibial nerves were ligated with 5.0 silk and a 1–2 mm section distal to the ligation was removed, leaving the sural nerve intact. The sham-operated mice underwent an identical procedure without nerve ligation or transection.
5.3.
Mechanical allodynia
All mice were habituated to the testing environment 2 days before baseline testing. Mice were individually placed in the plastic chamber on an elevated iron-mesh floor and allowed to acclimate for 20 min before testing. A series of von Frey filaments were presented perpendicularly to the lateral plantar surface of the paw for 5 s per filament, starting with filament 0.008 g. The 50% withdrawal threshold was calculated based on the last six stimuli, using the formula from Dixon (1980). The animals were tested 2 times before surgery to establish baseline responsiveness. Mechanical allodynia was tested on postoperative days 0.5, 1.0, 1.5, 3.0, and 7.0.
5.4. Cannulation surgery and intracerebroventricular injection Anesthetized mice were fixed in a stereotactic frame (Stoelting, WoodDale, IL, USA), the skin was incised longitudinally, and bregma was exposed. Stainless steel guide cannulae were bilaterally implanted to target the lateral cerebral ventricle (0.58 mm posterior to bregma, 1 mm lateral to midline, 2.5 mm deep from skull surface) and fixed into position with dental cement. Because intrathecal drug delivery and even vehicle injection may affect c-Fos expression (Luo et al., 1995), all three subgroups (Sham, SNI, and CSB6B) underwent identical procedures of cannulation surgery and drug delivery. After recovering from cannulation for 3 days, mice were microinjected with artificial cerebrospinal fluid (ACSF, 5 μL) or CSB6B (5 μg in 5 μL of ACSF) immediately after SNI surgery,
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followed by repeated administrations at 12-h intervals from postoperative day 1 to day 3.5. The development of mechanical allodynia was monitored before each drug administration. Another group of mice received CSB6B at 12-h intervals starting from postoperative day 7 and the effects of CSB6B on pain behavior were tested before each drug administration.
5.5.
Rotarod test
An accelerating rotarod was used to measure motor function. The mice were placed on a drum that rotated with a speed that increased from 0 to 25 rpm over 60 s, which was followed by an additional 240 s at the maximal speed, and their latency to falling was recorded. The mice were trained 4 times before baseline testing. Then the mice were i.c.v. injected with ACSF or CSB6B (5 μg) at 12-h intervals for 3 days, and tested before each drug administration.
5.6.
Tissue preparation
Mice were deeply anesthetized and perfused transcardially with saline solution followed by 4% paraformaldehyde in 0.1 mol/L phosphate buffer (PB) at pH 7.4 at 4 1C. Brains were carefully removed, further fixed in the same fixative for 24 h, and then soaked in 30% sucrose/PB for cryoprotection. By using a freezing microtome (Leica, Germany), coronal sections (20 μm) were collected within the following bregma intervals: 0.38 to 0.10 (for mPFC), 1.22 to 1.82 (for thalamus and amygdala), and 3.64 to 4.16 (for PAG) mm. Ten slices from each selected brain region were chosen from each animal. Mechanical stimulation of the hind paw was performed before perfusion to trigger c-Fos expression (Catheline et al., 1999).
5.7.
Immunohistochemistry
The sections were rinsed in 0.01 M phosphate buffered saline (PBS) three times before incubation in methanol containing 0.3% hydrogen peroxide for 30 min to block endogenous peroxidase activity. Antigen retrieval was then performed by microwaving in 0.01 M citrate buffer (pH 6.0). The sections were then treated with a blocking reagent containing goat serum for 30 min at room temperature (RT) and then incubated with mouse-anti-VGLUT2 antibody (1:2000; Abcam, Cambridge, UK) or rabbit-anti-Fos primary antibody (1:1500; Santa Cruz Biotechnology, Santa Cruz, CA) in PBS overnight at 4 1C. After incubation with secondary antibodies (biotinylated goat anti-mouse IgG and goat anti-rabbit IgG in PBS), the sections were successively treated with streptavidinhorseradish peroxidase (10 min), 3,30 -diaminobenzidine/H2O2 (10 min), and hematoxylin (1 min) at RT (all reagents from Immunoperoxidase Secondary Detection System, Millipore, MA, USA). Finally, the sections were dehydrated through an ascending series of ethanols, cleared with xylene, and coverslipped for microscopic observation. For antibody validation studies, the anti-VGLUT2 antibody was preincubated with a control protein (1:3000; 135-4p, synaptic systems, Göttingen, Germany).
5.8.
Synaptosome preparation
Synaptosomes were prepared from the whole thalamus of SNI and sham mice as previously described (Dunkley et al., 1986). On day 1 after SNI, the mice were deeply anesthetized and decapitated. The thalamus was then quickly removed and placed in ice-cold medium containing 320 mM sucrose (pH 7.4). After the tissue was homogenized, the homogenate was centrifuged at 3000g for 2 min at 4 1C, and the supernatant spun again at 14,500g for 12 min. Then, the pellet was gently resuspended, and 1 mL of this synaptosomal suspension was placed into 1.5 mL Percoll discontinuous gradients (3%, 10%, and 23%) containing 320 mM sucrose, 1 mM EDTA, and 0.25 mM d,l-dithiothreitol (pH 7.4). The suspension was centrifuged at 32,500g for 7 min at 4 1C, and synaptosomes sedimenting between the 10% and 23% Percoll bands were collected. These were diluted in 3 mL of HEPES buffer medium (HBM; 140 mM NaCl, 5 mM KCl, 5 mM NaHCO3, 1 mM MgCl2 6H2O, 1.2 mM Na2HPO4, 10 mM glucose, and 10 mM HEPES [pH 7.4]) and then centrifuged at 27,000 g for 10 min at 4 1C. The pellet was resuspended in HBM, and the protein content was determined using the Bicinchoninic Acid Protein Assay Kit (Applygen, Beijing, China). Finally, the synaptosomes were washed, stored on ice, and used within 4–6 h.
5.9.
Glutamate release assay
Glutamate release from thalamic synaptosomes was monitored by measuring the generation of fluorometrically detected NADPH (excitation and emission wavelengths of 340 and 460 nm, respectively) resulting from the oxidative deamination of released glutamate by exogenous glutamate dehydrogenase (Nicholls et al., 1987). The synaptosomal suspension (0.2 mg protein) or standard glutamate solution was added into a 96-well microplate with 100 μL in each well. After incubating for 3 min at 37 1C, 150 μL of a buffer solution containing 2 mM NADPþ, 50 U/mL glutamate dehydrogenase (Sigma-Aldrich, St. Louis, MO, USA), and 1 mM CaCl2 were added. After an additional 5 min of incubation, the baseline was tested for 100 s. Subsequently, 1 mM 4-aminopyridine (4AP) was added to stimulate glutamate release, and the increase of NADPH fluorescence was measured for 5 min. Data was read using a commercial plate reader (Perkin Elmer, Waltham, MA, USA). L-trans-2,4-Pyrrolidine dicarboxylic acid (L-trans-2,4-PDC, 50 mM) was added 10 min prior to the addition of 4-AP to inhibit EAATs. CSB6B at 2.5, 5, 10, 20, 40, 80, and 160 mM was added 30 min before the addition of 4-AP to inhibit VGLUTs.
5.10.
Microscopy
Following immunohistochemistry, brain sections were imaged with a Zeiss Axio Imager upright microscope equipped with a high-resolution digital camera. For VGLUT2 quantification, the integrated optical density (IOD) and area of interest (AOI) were measured with Image-Pro Plus 6.0 (Media Cybernetics, Bethesda, MD, USA), and the mean density was calculated as IOD/AOI in each image. For c-Fos quantification, the number of positive elements was also assessed by Image-Pro Plus 6.0.
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5.11.
Statistical analysis
Changes in protein expression, glutamate release, and behavioral manifestations among different groups were analyzed with one-way or two-way analysis of variance (ANOVA) followed by the Bonferroni post hoc test. All results are expressed as the mean7SEM.
Acknowledgments This work was supported by National Natural Science Foundation of China (81200850), Beijing Natural Science Foundation (7123224), National Science and Technology Major Project of China (2012ZX09301003-001).
Appendix A.
Supporting information
Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.brainres. 2015.08.010.
r e f e r e n c e s
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Research Report
Changes in VGLUT2 expression and function in pain-related supraspinal regions correlate with the pathogenesis of neuropathic pain in a mouse spared nerve injury model Zhi-Tong Wang, Gang Yun, Hong-Sheng Wang, Shou-Pu Yi, Rui-Bin Sun, Ze-Hui Gong State Key Laboratory of Toxicology and Medical Countermeasures, Beijing Institute of Pharmacology and Toxicology, 27 Taiping Road, Beijing 100850, People’s Republic of China
art i cle i nfo
ab st rac t
Article history:
Vesicular glutamate transporters (VGLUTs) control the storage and release of glutamate, which
Accepted 13 August 2015
plays a critical role in pain processing. The VGLUT2 isoform has been found to be densely
Available online 20 August 2015
distributed in the nociceptive pathways in supraspinal regions, and VGLUT2-deficient mice exhibit an attenuation of neuropathic pain; these results suggest a possible involvement of
Keywords:
VGLUT2 in neuropathic pain. To further examine this, we investigated the temporal changes in
Neuropathic pain
VGLUT2 expression in different brain regions as well as changes in glutamate release from
VGLUT2
thalamic synaptosomes in spared nerve injury (SNI) mice. We also investigated the effects of a
Supraspinal brain regions
VGLUT inhibitor, Chicago Sky Blue 6B (CSB6B), on pain behavior, c-Fos expression, and
Thalamus
depolarization-evoked glutamate release in SNI mice. Our results showed a significant elevation
Glutamate release
of VGLUT2 expression up to postoperative day 1 in the thalamus, periaqueductal gray, and
Chicago Sky Blue 6B
amygdala, followed by a return to control levels. Consistent with the changes in VGLUT2 expression, SNI enhanced depolarization-induced glutamate release from thalamic synaptosomes, while CSB6B treatment produced a concentration-dependent inhibition of glutamate release. Moreover, intracerebroventricular administration of CSB6B, at a dose that did not affect motor function, attenuated mechanical allodynia and c-Fos up-regulation in pain-related brain areas during the early stages of neuropathic pain development. These results demonstrate that changes in the expression of supraspinal VGLUT2 may be a new mechanism relevant to the induction of neuropathic pain after nerve injury that acts through an aggravation of glutamate imbalance. & 2015 Elsevier B.V. All rights reserved.
Abbreviations: VGLUT,
vesicular glutamate transporter; SNI,
amino acid transporter; PAG, PB,
phosphate-buffer; PBS,
periaqueductal gray; mPFC,
spared nerve injury; CSB6B,
medial prefrontal cortex; ACSF,
phosphate-buffered saline; 4-AP,
excitatory
4-aminopyridine; L-trans-2,4-PDC, L-trans-2,4-Pyrrolidine dicarboxylic
acid n Corresponding authors. Fax: þ86 10 68211656. E-mail addresses: [email protected] (G. Yu), [email protected] (R.-B. Su). http://dx.doi.org/10.1016/j.brainres.2015.08.010 0006-8993/& 2015 Elsevier B.V. All rights reserved.
Chicago Sky Blue 6B; EAAT, artificial cerebrospinal fluid;
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1.
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Introduction
Glutamatergic transmission and homeostasis are regulated by a glutamate transporter system that includes excitatory amino acid transporters (EAATs) and vesicular glutamate transporters (VGLUTs). EAATs remove glutamate from the neuronal synapse into glia and neurons to terminate excitatory signaling, whereas VGLUTs transport glutamate into vesicles prior to exocytotic release. Three distinct isoforms of VGLUTs have been identified (Ni et al., 1994; Takamori et al., 2001; Schafer et al., 2002), with VGLUT1 and VGLUT2 accounting for most of the presumed excitatory glutamatergic terminals in the central nervous system, whereas VGLUT3 is diffusely distributed in the brain, defining a discrete subpopulation of non-glutamatergic neurons (Kaneko and Fujiyama, 2002; Schafer et al., 2002). In adult rodents, VGLUT2 is most abundant in the diencephalon, brainstem, and spinal cord (Sakata-Haga et al., 2001; Kaneko and Fujiyama, 2002; Landry et al., 2004). The characteristic distribution pattern of VGLUT2 seems to be generally coincident with the nociceptive pathways, suggesting that VGLUT2 is involved in the signaling of pain. In addition, several studies in heterozygous mice have demonstrated that VGLUT2 deficiency results in attenuation or deletion of some neuropathic pain symptoms, unlike the effects of a VGLUT1 signaling impairment (Moechars et al., 2006; Leo et al., 2009). However, these results from genetically modified mice may be confounded by multiple factors, such as adaptation or compensation during development. Moreover, no study has investigated in wild-type animals possible time-dependent changes in VGLUTs in the central nervous system during the pathophysiological process of neuropathic pain development. The supraspinal regions are especially understudied in this connection. In the transmission of pain, nociceptive information from the spinal cord terminates in the thalamus, which is a key relay station for transmission to the cerebral cortex and periaqueductal gray (PAG), which are implicated in descending pain modulation (Millan, 2002). As to affect and cognition in pain processes, the amygdala modulates cortical functions associated with pain-induced mood disorders (Yalcin et al., 2014), while the medial prefrontal cortex (mPFC) is crucial for pain-related perception (Metz et al., 2009). Importantly, the thalamus, in which 90% of the excitatory synaptic response depends on VGLUT2, has been demonstrated to exhibit immediate reorganization after partial nerve ligation (Brüggemann et al., 2001) and abnormal discharge patterns in neuropathic pain models (Guilbaud et al., 1990). In addition, it has been reported that reduction or loss of VGLUT2 expression leads to reduction in the quantal size of glutamate release in thalamic neurons and is associated with attenuation of neuropathic pain in vivo (Moechars et al., 2006). In this study, we investigated temporal changes in VGLUT2 expression in pain-related brain areas in SNI mice by immunohistochemistry. In addition, Chicago Sky Blue 6B (CSB6B) was used as a pharmacological tool to examine whether inhibition of VGLUT activity would result in attenuation of thalamic glutamate release and pain behaviors. These results provide further evidence that VGLUT2 is associated with neuropathic pain.
2.
Results
2.1.
SNI-induced mechanical allodynia
The 25 mice that received SNI surgery were divided into 5 subgroups: SNI-0.5 d, SNI-1.0 d, SNI-1.5 d, SNI-3.0 d, and SNI7.0 d. We then validated that all the mice developed mechanical hypersensitivity to von Frey stimulation and no significant difference existed among these subgroups at the corresponding time points. Thus, only behavioral data in the SNI-7.0 d group are shown (Fig. 1). Repeated-measures two-way ANOVA showed significant effects of treatment (F [1,32]¼ 1025; po0.001) and a Bonferroni post hoc test revealed a significant decrease in the mechanical withdrawal threshold of the hind paws ipsilateral to the SNI surgery sides on postoperative days 0.5 (po0.001), 1.0 (po0.001), 1.5 (po0.001), 3.0 (po0.001), and 7.0 (po0.001).
2.2. Changes in VGLUT2 expression in pain-related brain areas after SNI The specificity of the VGLUT2 antibody has been validated by the pre-adsorption study (Supplementary Fig. 1). In the analyzed supraspinal areas, VGLUT2 immunostaining is punctuate, compatible with nerve terminals in the vicinity of neuronal cell bodies. Fig. 2 shows the changes in VGLUT2 expression at different times after SNI. Two-way ANOVA shows a significant effect of treatment in thalamus (F [1,40]¼ 20.81, po0.001) and PAG (F[1,40]¼ 4.67, po0.05). The Bonferroni post hoc test shows significant differences between sham and SNI mice in thalamus (po0.001) and PAG (po0.01) on postoperative day 1. Although two-way ANOVA does not show a significant effect of treatment in ipsilateral or contralateral amygdala, planned post hoc comparisons reveal a significant difference between sham and SNI mice on postoperative day 1 (contra, po0.01; ipsi,
Fig. 1 – Spared nerve injury (SNI) reduces paw withdrawal threshold. This was demonstrated by stimulating the sural nerve territory with a series of von Frey filaments. The mechanical threshold of SNI mice was significantly lower than that of the sham mice on postoperative days 0.5, 1.0, 1.5, 3.0, and 7.0, expressed as means7SEM. Note that the spacing of the time points is not to scale in this and subsequent figures n¼ 5; ***po0.001 compared with the sham control.
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po0.05). The up-regulation of VGLUT2 expression is transient and the level returns to baseline by 36 h (p40.05), remaining there for the rest of the experiment. In mPFC, there are no significant alterations in VGLUT2 staining, although a slight increase is observed on day 1.
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2.3. Effects of CSB6B on neuropathic pain and motor function in mice Fig. 3 shows the effects of CSB6B on SNI-induced neuropathic pain following different treatment schedules. Intracerebro-
Fig. 2 – Changes in VGLUT2 expression in supraspinal areas following spared nerve injury. The left panels show representative photomicrographs taken from SNI and sham mice (scale bar, 100 μm), showing an increase in VGLUT2 in SNI mice on postoperative day 1 (SNI-D1) within the analyzed brain regions, including thalamus (Thal), periaqueductal gray (PAG), amygdala (Amy), and medial prefrontal cortex (mPFC) compared with the sham control (Sham), followed by a return to sham control levels when examined on postoperative day 7 (SNI-D7). Plots on the right represent the quantitative labeling analysis expressed as relative mean density (n ¼5 for each group). Data are calculated as fold changes in mean density, and expressed as means7SEM; *po0.05; **po0.01; ***po0.001 compared with the sham control.
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ventricular administration of CSB6B (5 μg) at 12- h intervals starting from day 7 after SNI has no effect on established mechanical allodynia (Fig. 3B). The mice treated with CSB6B starting at the time of SNI, however, show a significant difference in the development of mechanical allodynia (F [1,84]¼ 8.17; po0.05). Bonferroni post hoc tests indicate that the pain threshold is significantly higher in CSB6B-treated mice than in their ACSF-treated counterparts on postoperative day 1.0 (0.6770.09 vs. 0.1470.04, po0.001). Nevertheless, considerable mechanical allodynia develops in CSB6B-treated mice, and no significant difference exists between the two groups from postoperative day 1.5–3.5 (p40.05, Fig. 3C). To exclude possible direct effects of CSB6B on motor function, we compared the rotarod performances of mice treated with ACSF and mice treated with CSB6B. No significant difference is seen between these two groups (F[1,56]¼ 0.09; p40.05; Fig. 3D), indicating that the apparent antinociceptive effects of CSB6B are not due to effects on motor function.
2.4. Effects of CSB6B on SNI-induced c-Fos expression in pain-related brain areas To further increase our confidence that CSB6B affects the degree of hyperalgesia after SNI surgery, the expression of
c-Fos in pain-related brain areas was tested by immunohistochemistry. Two-way ANOVA indicates significant effects of treatment in thalamus (F[2,36]¼9.70, po0.001), PAG (F[2,36]¼ 16.32, po0.001), contralateral amygdala (F[2,36]¼ 5.13, po0.05), ipsilateral amygdala (F[2,36]¼ 3.67, po0.05), contralateral mPFC (F[2,36]¼3.94, po0.05), and ipsilateral mPFC (F [2,36]¼ 6.57, po0.01) (Fig. 4). The Bonferroni post hoc test shows an SNI-induced up-regulation of c-Fos expression in thalamus (po0.05) and PAG (po0.001) on postoperative day 1, and in PAG (po0.01) and contralateral mPFC (po0.01) on postoperative day 1.5. Intracerebroventricular administration of CSB6B produces a tendency toward reduction in c-Fos expression in all areas analyzed and statistical significance is reached in thalamus on postoperative day 0.5 (po0.05) and in ipsilateral mPFC on postoperative day 1.5 (po0.05).
2.5. Effects of CSB6B on glutamate release in thalamic synaptosomes from SNI mice The depolarizing agent 4-AP releases neurotransmitter from the synaptic terminal in the presence of Ca2þ by inducing repetitive spontaneous Naþ-channel-dependent depolarizations, resembling the in vivo process of depolarization (Nicholls, 1998). Because EAAT expression was reported to change during SNI development, we added L-trans-2,4-PDC,
Fig. 3 – Effects of intracerebroventricular CSB6B on pain behavior and motor function. A: The distribution of CSB6B in the ventricular system after intracerebroventricular administration. After behavioral tests, mice were decapitated and the brains were removed to verify the placement of the cannula. Only the data from those animals with dispersion of the dye throughout the ventricles were used. B: Effects of CSB6B (5 μg in 5 μL ACSF, twice daily starting from postoperative day 7) on mechanical allodynia (n ¼5). C: Effects of CSB6B (5 μg, twice daily from postoperative day 1 to day 3.5 following an initial treatment immediately after the SNI) on mechanical allodynia (n ¼ 5 for control and n¼ 11 for CSB6B treatment). Data are expressed as means7SEM; ***po0.001 compared with the sham control. D: Effects of CSB6B on motor function. Mice injected i.c.v. with CSB6B (5 μg, twice daily) or ACSF show no differences in their ability to remain on an accelerating rotarod (n¼ 10). Data are expressed as means7SEM.
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Fig. 4 – Changes in numbers of c-Fos-immunoreactive cells in brain regions of SNI mice after CSB6B administration. The first three columns of panels show sections of thalamus and other pain processing areas from sham control and spared-nerve injured animals treated for 0.5 days with i.c.v. injections of ACSF or 5 μg of CSB6B (scale bar, 200 μm). Plots on the right represent the quantitative labeling analysis expressed as c-Fos-positive neurons/section (n ¼5 for each group). Data are expressed as mean7SEM; *po0.05 compared with the sham control; ♯po0.05 compared with the ACSF-treated group. which inhibits all five EAAT subtypes, to exclude the influence of plasma membrane reuptake on the concentration of glutamate in the incubation solution (Bridges et al., 1999). Fig. 5 shows the effects of SNI (on postoperative day 1) and CSB6B on 4-AP-evoked glutamate release in thalamic synaptosomes. One-way ANOVA shows a significant effect of treatment (F[3,16]¼ 70.15, po0.001). The Bonferroni post hoc test reveals a significant increase in glutamate release in SNI mice (po0.05) relative to sham-operated mice. Preincubation with CSB6B (20 μM) significantly reduces glutamate release in sham (po0.001) and SNI mice (po0.01, Fig. 5A). Fig. 5B shows
that the inhibitory effect of CSB6B is concentration-dependent, with an IC50 value of 25.7 μM.
3.
Discussion
To demonstrate the relation between VGLUT2 and neuropathic pain, we examined changes in VGLUT2 expression in pain-related brain areas after SNI surgery. The results showed an increase in VGLUT2 expression during an early postoperative stage followed by a return to control levels.
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Fig. 5 – CSB6B inhibits 4-AP-induced glutamate release from nerve terminals in mouse thalamus. A: Quantitative comparison of 4-AP-evoked glutamate release from thalamic synaptosomes 1 day after sham and SNI surgery. The presence of 20 μM CSB6B inhibits 4-AP-induced glutamate release in sham and SNI mice. Results are mean7SEM of 5 independent experiments; * po0.05; ***po0.001 compared with the sham control; ♯♯po0.01 compared with the SNI group. B: The concentration-response curve of the inhibitory action of CSB6B on 4-AP-evoked glutamate release. Results are mean7SEM of 5 independent experiments and the curve was fitted by nonlinear regression analysis with Prism 5.0 (GraphPad Software, Inc., San Diego, CA).
Correspondingly, inhibiting the activity of VGLUT2 reduced glutamate release from thalamic synaptosomes and attenuated the initial mechanical allodynia. Up-regulation of glutamatergic signaling has been proposed as a crucial mechanism of neuropathic pain. It has been demonstrated that nerve injury results in changes in the expression, distribution, and function of ionotropic and metabotropic glutamate receptors (Yogeeswari et al., 2009), as well as in the expression and uptake activity of EAATs (Sung et al., 2003), effects jointly contributing to changes in regional glutamate neurotransmission and pain behavior. More recently, a role for VGLUT2 in neuropathic pain has been suggested by studies of genetically modified mice. The heterozygous mice, which had a partial VGLUT2 deficiency, showed unchanged motor function, learning, memory, acute nociception, and inflammatory pain. However, they showed an absence of mechanical and cold allodynia after spared nerve injury (Moechars et al., 2006; Leo et al., 2009). In addition, conditioned knockout of VGLUT2 in DRG neurons attenuates physiological and pathological pain in mice (Liu et al., 2010; Scherrer et al., 2010). The present study further investigates the role of VGLUT2 in neuropathic pain, focusing on the expressional and functional changes in VGLUT2 in selected brain areas of wild-type SNI mice. The distribution of VGLUT2 supports the hypothesis that it plays a role in the processing of nociceptive signals. In the dorsal root ganglion, VGLUT2 is expressed by the majority of nociceptors (Scherrer et al., 2010). In the spinal cord, VGLUT2 is widely expressed, with the highest density in laminae I–II (Persson et al., 2006). In the brain, VGLUT2 mRNA and protein expression was reported to predominate in the thalamus, to be widely distributed in several PAG regions, and to be present in certain amygdaloid nuclei and cortical layers (Kaneko and Fujiyama, 2002; Wallen-Mackenzie et al., 2009;
Oka et al., 2012). In addition, a study in mutant mice shows that VGLUT2-mediated neurotransmission also participates in the neuronal circuitry of higher brain functions, despite its limited expression in forebrain areas (Wallen-Mackenzie et al., 2009). Consistent with previous studies, we observed significant VGLUT2 staining in the above pain-related areas and VGLUT2 seems to be expressed mainly in afferent nerves arising from lower central nervous system (Graziano et al., 2008). Moreover, we found that VGLUT2 expression showed an increase in thalamus, PAG, and amygdala, with a maximum extent of up-regulation observed in thalamus on postoperative day 1, suggesting a molecular rearrangement of excitatory transmission in these brain areas after peripheral nerve injury (Marcello et al., 2013). Although the underlying mechanisms remains to be determined, we speculate that the up-regulation of VGLUT2 may be related to changes in brain-derived neurotrophic factor (BDNF), because BDNF has been reported to increase VGLUT expression in cultured neurons (Melo et al., 2013) and to be upregulated in the central nervous system starting from day 1 after SNI (Zhou et al., 2014). We also found that the up-regulation of VGLUT2 lasted for a very short time, and was followed by a return to control levels, which may be due to inactivation of the TrkB signaling pathway (Melo et al., 2013). This may be a compensatory effect preventing over-activation of glutamatergic neurotransmission. It has been demonstrated that the expression of EAATs is altered after nerve injury in the spinal cord and some brain structures, and contributes to a disturbance of glutamate clearance and to neuropathic pain (Sung et al., 2003; Marcello et al., 2013). However, the changes in VGLUT2 expression in neuropathic pain and their relationship to glutamate release are not thoroughly understood. Considering its crucial role in pain sensation, the thalamus was selected to demonstrate
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the functional consequence of VGLUT2 up-regulation. We observed an increase in depolarization-induced glutamate release from synaptosomes prepared from thalamic tissue one day after SNI. This finding was in agreement with a study in genetically modified mice demonstrating that a deficiency of VGLUT2 protein resulted in abolition of neuropathic pain and a reduction in miniature EPSC amplitudes, indicating that VGLUT protein levels determine the glutamate content loaded into single vesicles and the size of the subsequent quantal release (Moechars et al., 2006). Our findings further confirm the role of VGLUT2 in neuropathic pain, by demonstrating that the SNI-induced increase in VGLUT2 expression led to an increase of glutamate release in the thalamus and thus contributed to the development of pain behavior. CSB6B is an azo dye that potently and selectively inhibits the function of VGLUTs without affecting plasma membrane transporters (Thompson et al., 2005; Roseth et al., 1995). Previously, we observed that i.c.v. administration of CSB6B did not affect the acute phasic pain but attenuated tonic inflammatory pain in mouse models (Yu et al., 2013). In this study, behavioral assays further showed that inhibiting the function of VGLUTs significantly reduced the development of mechanical allodynia in the initial period of neuropathic pain. The inferred antinociceptive effects of CSB6B were corroborated by changes in expression of c-Fos, a painrelated biochemical marker (Bullitt, 1990; Munglani et al., 1999), and by the absence of a direct influence on motor function. The inhibitory effects of CSB6B on thalamic glutamate release that we observed provide a plausible mechanism for the behavioral results. Although the inhibitory effect of CSB6B is not specific to one VGLUT isoform, we speculate that it exerted its effects by inhibiting the enhancement of glutamate neurotransmission caused by VGLUT2 up-regulation, as VGLUT2 is mainly expressed in pain-related brain areas and in particular 90% of thalamic excitatory synaptic response depends on VGLUT2. Our findings are in agreement with results from genetically engineered mice (Moechars et al., 2006; Leo et al., 2009). The effects of CSB6B seem to be related to upregulated VGLUT2 expression during the early stage of neuropathic pain, and the subsequent decay of its effects are consistent with the subsequent expression decreases in VGLUT2. The lack of effects of CSB6B along with normalized VGLUT2 expression during the later stages of neuropathic pain suggest that VGLUT2 mainly participates in the initiation of neuropathic pain, in advance of a variety of other factors dynamically changing during the pain development process (Latremoliere and Woolf, 2009).
4.
Conclusions
We have demonstrated that SNI can induce time-dependent changes in VGLUT2 expression in pain-related brain areas and that up-regulation of VGLUT2 increases presynaptic glutamate release in the thalamus. CSB6B produced antinociceptive effects on neuropathic pain in its initial stages, simultaneously decreasing depolarization-evoked glutamate release from thalamic synaptosomes. Therefore, the findings suggest a possible correlativity between VGLUT2 and neuropathic pain in supraspinal regions and a possibility of treating
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neuropathic pain by inhibiting VGLUTs to reduce glutamate availability.
5.
Experimental procedures
5.1.
Animals
All experiments were approved by the local ethical committee and the Institutional Review Committee on Animal Care, and were conducted in accordance with their guidelines for the use of experimental animals. Eight-week-old C57BL/6 male mice (Beijing Animal Center, Beijing, China) were housed at constant temperature (2172 1C) on a 12-h light/ dark cycle with free access to food and water.
5.2.
Spared nerve injury (SNI) surgery
Under pentobarbital sodium (40 mg/kg, i.p.) anesthesia, mice underwent the following surgery according to the method of Decosterd and Woolf (2000). The three terminal branches of the sciatic nerve (the common peroneal, tibial, and sural nerves) were exposed after skin and muscle incision. Both the common peroneal and tibial nerves were ligated with 5.0 silk and a 1–2 mm section distal to the ligation was removed, leaving the sural nerve intact. The sham-operated mice underwent an identical procedure without nerve ligation or transection.
5.3.
Mechanical allodynia
All mice were habituated to the testing environment 2 days before baseline testing. Mice were individually placed in the plastic chamber on an elevated iron-mesh floor and allowed to acclimate for 20 min before testing. A series of von Frey filaments were presented perpendicularly to the lateral plantar surface of the paw for 5 s per filament, starting with filament 0.008 g. The 50% withdrawal threshold was calculated based on the last six stimuli, using the formula from Dixon (1980). The animals were tested 2 times before surgery to establish baseline responsiveness. Mechanical allodynia was tested on postoperative days 0.5, 1.0, 1.5, 3.0, and 7.0.
5.4. Cannulation surgery and intracerebroventricular injection Anesthetized mice were fixed in a stereotactic frame (Stoelting, WoodDale, IL, USA), the skin was incised longitudinally, and bregma was exposed. Stainless steel guide cannulae were bilaterally implanted to target the lateral cerebral ventricle (0.58 mm posterior to bregma, 1 mm lateral to midline, 2.5 mm deep from skull surface) and fixed into position with dental cement. Because intrathecal drug delivery and even vehicle injection may affect c-Fos expression (Luo et al., 1995), all three subgroups (Sham, SNI, and CSB6B) underwent identical procedures of cannulation surgery and drug delivery. After recovering from cannulation for 3 days, mice were microinjected with artificial cerebrospinal fluid (ACSF, 5 μL) or CSB6B (5 μg in 5 μL of ACSF) immediately after SNI surgery,
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followed by repeated administrations at 12-h intervals from postoperative day 1 to day 3.5. The development of mechanical allodynia was monitored before each drug administration. Another group of mice received CSB6B at 12-h intervals starting from postoperative day 7 and the effects of CSB6B on pain behavior were tested before each drug administration.
5.5.
Rotarod test
An accelerating rotarod was used to measure motor function. The mice were placed on a drum that rotated with a speed that increased from 0 to 25 rpm over 60 s, which was followed by an additional 240 s at the maximal speed, and their latency to falling was recorded. The mice were trained 4 times before baseline testing. Then the mice were i.c.v. injected with ACSF or CSB6B (5 μg) at 12-h intervals for 3 days, and tested before each drug administration.
5.6.
Tissue preparation
Mice were deeply anesthetized and perfused transcardially with saline solution followed by 4% paraformaldehyde in 0.1 mol/L phosphate buffer (PB) at pH 7.4 at 4 1C. Brains were carefully removed, further fixed in the same fixative for 24 h, and then soaked in 30% sucrose/PB for cryoprotection. By using a freezing microtome (Leica, Germany), coronal sections (20 μm) were collected within the following bregma intervals: 0.38 to 0.10 (for mPFC), 1.22 to 1.82 (for thalamus and amygdala), and 3.64 to 4.16 (for PAG) mm. Ten slices from each selected brain region were chosen from each animal. Mechanical stimulation of the hind paw was performed before perfusion to trigger c-Fos expression (Catheline et al., 1999).
5.7.
Immunohistochemistry
The sections were rinsed in 0.01 M phosphate buffered saline (PBS) three times before incubation in methanol containing 0.3% hydrogen peroxide for 30 min to block endogenous peroxidase activity. Antigen retrieval was then performed by microwaving in 0.01 M citrate buffer (pH 6.0). The sections were then treated with a blocking reagent containing goat serum for 30 min at room temperature (RT) and then incubated with mouse-anti-VGLUT2 antibody (1:2000; Abcam, Cambridge, UK) or rabbit-anti-Fos primary antibody (1:1500; Santa Cruz Biotechnology, Santa Cruz, CA) in PBS overnight at 4 1C. After incubation with secondary antibodies (biotinylated goat anti-mouse IgG and goat anti-rabbit IgG in PBS), the sections were successively treated with streptavidinhorseradish peroxidase (10 min), 3,30 -diaminobenzidine/H2O2 (10 min), and hematoxylin (1 min) at RT (all reagents from Immunoperoxidase Secondary Detection System, Millipore, MA, USA). Finally, the sections were dehydrated through an ascending series of ethanols, cleared with xylene, and coverslipped for microscopic observation. For antibody validation studies, the anti-VGLUT2 antibody was preincubated with a control protein (1:3000; 135-4p, synaptic systems, Göttingen, Germany).
5.8.
Synaptosome preparation
Synaptosomes were prepared from the whole thalamus of SNI and sham mice as previously described (Dunkley et al., 1986). On day 1 after SNI, the mice were deeply anesthetized and decapitated. The thalamus was then quickly removed and placed in ice-cold medium containing 320 mM sucrose (pH 7.4). After the tissue was homogenized, the homogenate was centrifuged at 3000g for 2 min at 4 1C, and the supernatant spun again at 14,500g for 12 min. Then, the pellet was gently resuspended, and 1 mL of this synaptosomal suspension was placed into 1.5 mL Percoll discontinuous gradients (3%, 10%, and 23%) containing 320 mM sucrose, 1 mM EDTA, and 0.25 mM d,l-dithiothreitol (pH 7.4). The suspension was centrifuged at 32,500g for 7 min at 4 1C, and synaptosomes sedimenting between the 10% and 23% Percoll bands were collected. These were diluted in 3 mL of HEPES buffer medium (HBM; 140 mM NaCl, 5 mM KCl, 5 mM NaHCO3, 1 mM MgCl2 6H2O, 1.2 mM Na2HPO4, 10 mM glucose, and 10 mM HEPES [pH 7.4]) and then centrifuged at 27,000 g for 10 min at 4 1C. The pellet was resuspended in HBM, and the protein content was determined using the Bicinchoninic Acid Protein Assay Kit (Applygen, Beijing, China). Finally, the synaptosomes were washed, stored on ice, and used within 4–6 h.
5.9.
Glutamate release assay
Glutamate release from thalamic synaptosomes was monitored by measuring the generation of fluorometrically detected NADPH (excitation and emission wavelengths of 340 and 460 nm, respectively) resulting from the oxidative deamination of released glutamate by exogenous glutamate dehydrogenase (Nicholls et al., 1987). The synaptosomal suspension (0.2 mg protein) or standard glutamate solution was added into a 96-well microplate with 100 μL in each well. After incubating for 3 min at 37 1C, 150 μL of a buffer solution containing 2 mM NADPþ, 50 U/mL glutamate dehydrogenase (Sigma-Aldrich, St. Louis, MO, USA), and 1 mM CaCl2 were added. After an additional 5 min of incubation, the baseline was tested for 100 s. Subsequently, 1 mM 4-aminopyridine (4AP) was added to stimulate glutamate release, and the increase of NADPH fluorescence was measured for 5 min. Data was read using a commercial plate reader (Perkin Elmer, Waltham, MA, USA). L-trans-2,4-Pyrrolidine dicarboxylic acid (L-trans-2,4-PDC, 50 mM) was added 10 min prior to the addition of 4-AP to inhibit EAATs. CSB6B at 2.5, 5, 10, 20, 40, 80, and 160 mM was added 30 min before the addition of 4-AP to inhibit VGLUTs.
5.10.
Microscopy
Following immunohistochemistry, brain sections were imaged with a Zeiss Axio Imager upright microscope equipped with a high-resolution digital camera. For VGLUT2 quantification, the integrated optical density (IOD) and area of interest (AOI) were measured with Image-Pro Plus 6.0 (Media Cybernetics, Bethesda, MD, USA), and the mean density was calculated as IOD/AOI in each image. For c-Fos quantification, the number of positive elements was also assessed by Image-Pro Plus 6.0.
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5.11.
Statistical analysis
Changes in protein expression, glutamate release, and behavioral manifestations among different groups were analyzed with one-way or two-way analysis of variance (ANOVA) followed by the Bonferroni post hoc test. All results are expressed as the mean7SEM.
Acknowledgments This work was supported by National Natural Science Foundation of China (81200850), Beijing Natural Science Foundation (7123224), National Science and Technology Major Project of China (2012ZX09301003-001).
Appendix A.
Supporting information
Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.brainres. 2015.08.010.
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