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Upregulation of glutamatergic transmission in anterior cingulate cortex in the diabetic rats with neuropathic pain
Neuroscience Letters 568 (2014) 29–34
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Upregulation of glutamatergic transmission in anterior cingulate cortex in the diabetic rats with neuropathic pain Weifang Li a , Peng Wang b , Hua Li a,∗ a b
Department of Geriatric Endocrinology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan 450001, China Department of Epidemiology, College of Public Health, Zhengzhou University, Zhengzhou, Henan 450001, China
h i g h l i g h t s • STZ induced hyperglycemia, thermal hyperalgesia and tactile allodynia in rats. • Enhanced glutamatergic transmission in the ACC neurons of the modeled rats. • Increased PKM phosphorylation in ACC of the modeled rats.
a r t i c l e
i n f o
Article history: Received 26 January 2014 Received in revised form 21 February 2014 Accepted 7 March 2014 Keywords: Diabetic neuropathic pain Anterior cingulate cortex Glutamatergic transmission Protein kinase M
a b s t r a c t Peripheral neuropathic pain is a common complication in the diabetic patients, and the underlying central mechanism remains unclear. Forebrain anterior cingulate cortex (ACC) is critically involved in the supraspinal perception of physical and affective components of noxious stimulus and pain modulation. Excitatory glutamatergic transmission in the ACC extensively contributed to the maintenance of negative affective component of chronic pain. The present study examined the adaptation of glutamatergic transmission in the ACC in rats with diabetic neuropathic pain. Injection with streptozotocin (STZ) induced hyperglycemia, thermal hyperalgesia and mechanical allodynia in the rats. In these rats, significant enhanced basal glutamatergic transmission was observed in the ACC neurons. The increased presynaptic glutamate release and enhanced conductance of postsynaptic glutamate receptors were also observed in the ACC neurons of these modeled rats. Increased phosphorylation of PKM, but not the expression of total PKM, was also observed in the ACC. Microinjection of PKM inhibitor ZIP into ACC attenuated the upregulation of glutamate transmission and painful behaviors in STZ-injected rats. These results revealed a substantial central sensitization in the ACC neurons in the rodents with diabetic neuropathic pain, which may partially underlie the negative affective components of patients with diabetic neuropathic pain. © 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Peripheral neuropathic pain is a common complication in the diabetic patients. While a number of studies demonstrate that significant plasticity occurs in the spinal nocisponsive neurons, emerging evidences indicate significant adaptation in supraspinal brain regions pertinent to the processing of pain information. Forebrain anterior cingulate cortex (ACC), a brain region processing the cognitive and emotional functions of the brain, is critically involved in the supraspinal perception of noxious stimulus and pain modulation [27]. Previous anatomical, behavioral and physiological evidences suggest that neuronal circuit within and among ACC and
∗ Corresponding author. Tel.: +86 371 66913114. E-mail address: [email protected] (H. Li). http://dx.doi.org/10.1016/j.neulet.2014.03.038 0304-3940/© 2014 Elsevier Ireland Ltd. All rights reserved.
other brain regions (e.g., amygdala) processes information relating to the affective and/or physical components of peripheral painful sensation [8,13]. Peripheral noxious stimuli significantly increased the excitability of anterior cingulate cortex neurons [19], and local lesions of the anterior cingulate cortex reduced acute nociceptive responses and injury-related aversive behaviors in rodent model of chronic pain [9,28]. It was also documented that significant upregulation of excitatory glutamatergic transmission was observed in the ACC neurons, which critically contributing to the transition from acute nociceptive sensation to persisted pain [27,28] as well as the perception of negative components of pain. While increasing evidences demonstrate the significant adaptation of peripheral sensory neurons and dorsal horn neurons in the diabetic neuropathic pain, the character of central sensitization in nocisponsive supraspinal brain regions, such as ACC, remain unexplored.
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Accumulating evidences demonstrate the significant adaptation of plasticity of central neurocircuits, either excitatory or inhibitory, occurs in the physiological, such as learning and memory, and pathological conditions such as drug abuse [20] and chronic pain [27]. Among these, enhanced glutamatergic transmission in spinal dorsal horn and supraspinal regions has been extensively recognized for its critical role in the transition of from acute to chronic pain [16]. Peripheral inflammation and nerve injury induce the increase of presynaptic glutamate release [25] and membrane trafficking of AMPA receptor subunit GluR1 [2] in the ACC neurons. Currently, it remains unclear whether significant adaptation of glutamatergic transmission occurs in the ACC neurons in the setting of diabetic neuropathic pain. Activation of several intracellular pathways substantially modulates the synaptic plasticity in the central neurons in the setting of chronic pain [28]. Recent studies showed that an atypical PKC, PKM, critically contributed to sensitization of glutamatergic transmission in spinal and supraspinal nocisponsive neurons in the condition of peripheral nerve injury [11,13]. In the present study, we also study the functional change of PKM in the ACC of the rodent model of diabetic neuropathic pain. 2. Materials and methods 2.1. Animal model of streptozotocin-induced diabetic rats Male Sprague-Dawley rats (180–220 g) were purchased from the institutional center of experimental animals. All experimental protocols were approved by the Institutional Animal Care and Use Committee, and were conducted in accordance with National Institutes of Health guidelines.
Diabetes was induced by a single intraperitoneal injection of streptozotocin (STZ; 60 mg/kg; Sigma–Aldrich) freshly dissolved in 0.9% sterile saline as previously described [12]. Blood glucose levels were assayed using ACCU-CHEK test strips (Roche Diagnostics, Indianapolis) at 2 and 6 weeks after STZ administration, and the weights were measured weekly. Age-matched rats injected with vehicle were used as controls. 2.2. Cannula implantation and behavioral test A 30 gauge stainless steel cannula with a 33 gauge stylet plug was bilaterally implanted 0.5 mm above the ACC [1]. The rats were allowed one week recovery prior to further study. CNQX (10 nmol/0.5 l per side), ZIP (10 nmol/0.5 l per side), or vehicle was daily injected into the ACC 3 days prior to the behavioral test at week 6. Mechanical allodynia and thermal hyperalgesia were assessed in rats to evaluate painful behavior. Mechanical sensitivity was assessed by using a series of von Frey filaments with logarithmic incremental stiffness (Stoelting Co., Wood Dale, IL), and 50% probability paw withdrawal thresholds were calculated with the up–down method [14]. The thermal nociceptive thresholds were measured using radiant heat [6], and the time for the rat to remove the paw from the thermal stimulus was electronically recorded as the paw withdrawal latency (PWL). 2.3. Whole-cell patch clamp recordings in ACC slices The electrophysiological recording was performed in the rats with behavioral tests. The coronal brain slices (300 m) containing
Fig. 1. STZ induced hyperglycemia, thermal hyperalgesia and mechanical allodynia in the rats. (A) Significantly increased blood glucose levels were observed in the rats at 2 and 6 weeks after STZ injection (n = 19–22 rats in each group). (B) Significant lower body weight was observed in the STZ-injected rats (n = 19–22 rats in each group). Time course of hindpaw withdrawal latency response to radiant thermal stimulation (C) and paw withdrawal threshold response to mechanical stimulation (D) in the rats treated with STZ or vehicle (n = 11 rats in each group). * P < 0.05, ** P < 0.01; *** P < 0.001.
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the ACC were obtained, and the whole-cell patch clamp recordings was performed on the ACC slices after 1-h recovery as previously reported [3]. Excitatory postsynaptic currents (EPSCs) were recorded from layer II/III neurons with a electrical stimulation (0.25 ms, 0.05–0.3 mA) in layer V in ACC. The membrane potential was held at −70 mV throughout the experiment. The glutamate synaptic strength was investigated with three graded stimulus intensities, and the input (stimulus intensity)-out (EPSC amplitude) response was plotted accordingly. For paradigm of paired-pulse ratios (PPR), a pair of EPSCs was evoked by two stimuli with an interval of 40, 60 and 80 ms. Four PPRs were averaged to obtain a mean PPR to minimum the variances among consecutive events. 2.4. Protein extraction and immunoblotting The rostral ACC tissues were punched from stereotaxically identified 2 mm thick slices (bregma +3.7 to +1.7) from the rats and proceeded to immunoblotting as described previously [11]. Primary antibodies against phospho (Thr 410) – PKM (Cell Signaling Technology, 1:1000), PKM (Cell Signaling Technology, 1:1000), -actin (Santa Cruz Biotechnology, 1: 1000) and appropriate secondary antibodies were used. The immunoreactivity was detected using enhanced chemiluminescence (ECL Advance Kit; Amersham Biosciences). The immunoreactivity of target proteins was normalized to that of -actin. 2.5. Drug and data analysis 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX), 2-amino-5phos-phonopentanoic acid (AP-5), ␣-amino-3-hydroxy-5-methyl4-isoxazolepropionic acid (AMPA), bicuculline and all other drugs were purchased from Sigma–Aldrich or Tocris, and were applied through the perfusion solution. Results were analyzed by paired or unpaired t-test, or two-way ANOVA followed by post hoc test. All data are expressed as mean ± S.E.M. P < 0.05 was considered statistically significant. 3. Results 3.1. Blood glucose levels and body weights of the experimental groups As shown in Fig. 1, the rats treated with STZ consistently showed significantly higher blood glucose levels (429 ± 27 mg/dl, n = 22 rats) compared to the rats treated with vehicle (142 ± 15 mg/dl, n = 19 rats) at 2 weeks after STZ injection (Fig. 1A). Such hyperglycemia was observed in all of the rats at 6 weeks after STZ injection (Fig. 1A). Meanwhile, the body weights of STZ-treated rats were significantly lower than those of vehicle-treated rats during 6 weeks of observation (Fig. 1B). These observations indicated that all of the rats that received STZ became diabetic, in accordance with previous reports [12]. 3.2. Significant thermal hyperalgesia and mechanical allodynia in the STZ-treated rats Consistent with previous reports [12,21], the STZ-injected rats showed significantly decreased hindpaw withdrawal latencies response to the radiant thermal stimuli (Fig. 1C), which obviously manifested at 2 weeks after the STZ injection, and maintained through at least 6 weeks after STZ injection. Similarly, significantly lower paw withdrawal thresholds response to mechanical stimuli were also observed from 2 weeks through at least 6 weeks after injection with STZ (Fig. 1D). These results confirmed the thermal hyperalgesia and mechanical allodynia in the rats injected with STZ.
Fig. 2. STZ increased glutamatergic strength in the ACC neurons. Significantly enhanced input (stimuli intensity)–output (EPSC amplitude) was observed in the ACC neurons of STZ-treated rats, which was attenuated by the microinjection of ZIP, a selective PKM inhibitor, into the ACC. n = 9–11 neurons per group. * P < 0.05; ** P < 0.01.
3.3. Significantly enhanced glutamatergic transmission in the ACC in the STZ-treated rats We then examined whether significant adaptation of glutamatergic transmission occurred in the ACC. The EPSCs were recorded in the presence of bicuculline (30 M) by the electric stimuli with three graded intensities. The evoked EPSCs were almost completely abolished by the application of AP-V (10 M) and CNQX (10 M) (146.2 ± 8.7 pA vs. 9.7 ± 3.6 pA, n = 9 neurons, P < 0.01), confirming the glutamatergic components. As shown in Fig. 2, significant left shift of the input–output response of the evoked EPSCs was observed in ACC neurons in the STZ-treated rats. Meanwhile, microinjection of CNQX (10 nmol/side) significantly attenuated the thermal hyperalgesia and mechanical allodynia in the rats injected with STZ (Fig. 3C and D). These suggested the enhanced glutamatergic strength in the ACC neurons of STZ-treated rats with neuropathic pain. 3.4. Significantly enhanced presynaptic glutamate release in the ACC in the STZ-treated rats Upregulation of glutamatergic transmission in the central neurons may result from the enhanced presynaptic glutamate release and/or the redistribution of the postsynaptic glutamate receptor subunits. Firstly, the paradigm of paired-pulse ratio (PPR) was applied to determine whether enhanced presynaptic glutamate release existed in the ACC neurons in the STZ-treated rats. As shown in Fig. 3A, the ACC neurons in STZ-injected rats had a PPR (inter-stimulation interval of 40 ms) of 1.19 ± 0.07 (n = 14 neurons), which was significantly less than that in the vehicle-treated rats (1.44 ± 0.08, n = 12 neurons, P < 0.05). The decreased PPR was consistently found at the stimuli with three different intervals (40 ms,
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Fig. 3. STZ increased presynaptic glutamate release and conductance of postsynaptic AMPA receptors in the ACC neurons. Significantly decreased PPR of evoked EPSCs was observed in the ACC neurons of STZ-treated rats at three different inter-stimuli intervals (A, n = 12 and 14 neurons in each group). Exogenous AMPA (1 M) elicited significantly stronger inward currents in the ACC neurons of the STZ-treated rats (B, n = 14 and 17 neurons in each group). Microinjection of CNQX (10 nmol/side) into the ACC significantly attenuated the thermal hyperalgesia and mechanical allodynia in the rats injected with STZ (C and D, n = 8–9 rats per group). Vehicle vs. STZ: * P < 0.05; ** P < 0.01; STZ vs. STZ + CNQX: ## P < 0.01.
60 ms, and 80 ms). These results suggested an enhanced presynaptic glutamate release in the ACC neurons in the STZ-treated rats. 3.5. Significantly enhanced inward current evoked by AMPA To determine whether adaptation of postsynaptic glutamate receptor exists in the ACC neurons in STZ-treated rats, exogenous AMPA (1 M) was perfused, and the inward current was recorded. As shown in Fig. 3B, AMPA (1 M) induced a significant larger inward current in the ACC neurons of the STZ-treated rats (135.4 ± 10.3 pA, n = 17 neurons, P < 0.01) than that in the vehicle-treated rats (89.6 ± 7.5 pA, n = 14 neurons). This suggested an adaptation of postsynaptic glutamate receptor in ACC neurons of the STZ-treated rats.
As shown in Fig. 4A, while the expression of total PKM remained unchanged, the phosphorylation of PKM (Thr 410) was significantly increased in the ACC tissues in the STZ-treated rats (n = 7–8 rats in each group). Bilateral microinjections of ZIP (10 nmol/side for 3 days), a selective cell-permeable PKM inhibitor, into the ACC significantly attenuated the upregulation of glutamatergic transmission (Fig. 2), and reduced the thermal hyperalgesia and mechanical allodynia in the rats injected with STZ (Fig. 4B and C). This implied, considering the functional significance of PKM in synaptic plasticity, that an upregulation of PKM signaling may account for the enhanced glutamatergic transmission in the ACC neurons of STZ-treated rats. 4. Discussion
3.6. Significantly increased expression of PKM It was well documented that upregulation of PKM signaling significantly contributed to the maintenance of synaptic plasticity in the brain neurons, and was critically involved in the central sensitization in several models of chronic pain [11,13]. Here immunoblotting technique was applied to detect the expression of PKM and its phosphorylation in the ACC tissues of the diabetic rats.
In the present study, we identified the enhanced glutamatergic transmission, via increased presynaptic glutamate release and enhanced conductance of postsynaptic AMPA receptor, in the ACC neurons in the diabetic neurons. We also found an increased phosphorylation of PKM, an atypical PKC, in the ACC, which possibly accounted for the enhanced glutamatergic transmission in these modeled rats. To our knowledge, this is the first investigation to
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Fig. 4. Significantly increased phosphorylation of PKM (Thr 410) was observed in the ACC tissues in the STZ-treated rats, while the expression of total PKM remained unchanged (n = 7 and 8 rats in each group). Microinjection of ZIP (10 nmol/side) into the ACC significantly attenuated the thermal hyperalgesia and mechanical allodynia in the rats injected with STZ (B and C, n = 8–9 rats per group). Vehicle vs. STZ: ** P < 0.01; STZ vs. STZ + ZIP: ## P < 0.01.
elucidate the central sensitization and the possible involvement of PKM in the in the anterior cingulate cortex, the key brain region for pain and emotion modulation, in the diabetic rats with neuropathic pain. Currently, a number of evidences demonstrated that the forebrain anterior cingulate cortex, a brain region processing the cognitive and emotional function, was critically involved in the perception of physical [27,28] and affective [5,13] components of pain. Significant depolarized membrane potential, lower firing threshold, lower input resistance and increased spike frequency were previously observed in the ACC of the rats with peripheral persistent painful stimuli [4]. Spinal injury induced the loss of the gray matter volume [23], and continuous inhibition of painful stimuli was associated with the increased gray matter in ACC in the patients with chronic pain [17]. In the present study, an enhanced glutamatergic transmission was, for the first time, revealed in the ACC neurons of the rats with diabetic neuropathic pain, which was attributable to the enhanced presynaptic glutamate release, indicated by decreased paired-pulse ratio of evoked EPSCs, and increased conductance of postsynaptic glutamate receptors, indicated by increased inward current evoked by exogenous AMPA (1 M). Previous studies ever found the similar enhanced glutamatergic transmission, along with the reduced inhibitory transmission, was observed in the ACC neurons in the rats with persistent painful stimuli [4], and suppression of the glutamatergic transmission in ACC neurons significantly attenuated the pain-evoked place escape/avoidance in the rat with chronic nerve constriction injury [5]. Significant increased presynaptic glutamate release and postsynaptic AMPA receptor subunit GluR1 membrane trafficking were ever reported in the ACC neurons of the
rats with chronic pain induced by peripheral inflammation [26] or nerve injury [22,25]. Similar adaptation of glutamatergic transmission in ACC was also observed in the rodent model with visceral pain [24], or bone-cancer pain [3]. Hence, it is postulated that similar redistribution of receptor subunits likely account for the increased conductance of postsynaptic AMPA receptors in the ACC of the diabetic rats. Taken together, these findings suggested that enhanced glutamate transmission in ACC neurons was a fundamental longterm plasticity during chronic pain, which would be responsible for the affective response to chronic pain and the aversion to noxious stimulus. PKM is an atypical PKC, which does not respond to either intracellular Ca2+ or diacylglycerol but is regulated by protein–protein interactions and potentially membrane lipid composition [7]. The expression of PKM is primarily restricted to neurons [15]. The character of autonomous activity suggests that PKM is an important player in LTP maintenance. Actually, strong synaptic stimulation, while inducing LTP, is associated with phosphorylation of PKM [10], and inhibition of PKM activity by a pseudosubstrate inhibitor, ZIP, significantly abolished the maintenance of LTP in the cortex [18]. Furthermore, peripheral nerve injury induced an early increase in PKM expression and a persistent increase in PKM phosphorylation in the ACC in the mice, and suppression of PKM activity reduced the upregulation of glutamatergic transmission in ACC neurons, as well as the mechanical allodynia, in the mice with neuropathic pain [13]. Here, a significant increased phosphorylation of PKM, but not the expression of total PKM, was observed in the ACC tissues, and inhibition of PKM activity alleviated the upregulation of glutamate transmission and pain behaviors in the rats with diabetic neuropathic pain. These results suggested that
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the increased PKM activity may underlie the upregulation of glutamatergic transmission in ACC neurons of the rats with diabetic neuropathic pain. In conclusion, the present study demonstrated a significantly enhanced glutamatergic transmission, which was attributable to the increased presynaptic glutamate release and enhanced conductance of postsynaptic glutamate receptors, in the ACC neurons of the rats with diabetic neuropathic pain. An increased phosphorylation of PKM may account for this upregulation of glutamatergic transmission in the molded rats. This investigation may partially elucidate the cellular mechanism for the negative affective components in the patients with diabetic neuropathic pain. References [1] H. Cao, Y.J. Gao, W.H. Ren, T.T. Li, K.Z. Duan, Y.H. Cui, X.H. Cao, Z.Q. Zhao, R.R. Ji, Y.Q. Zhang, Activation of extracellular signal-regulated kinase in the anterior cingulate cortex contributes to the induction and expression of affective pain, Journal of Neuroscience: Official Journal of the Society for Neuroscience 29 (2009) 3307–3321. [2] X.Y. Cao, H. Xu, L.J. Wu, X.Y. Li, T. Chen, M. Zhuo, Characterization of intrinsic properties of cingulate pyramidal neurons in adult mice after nerve injury, Molecular Pain 5 (2009) 73. [3] C.S. Chiou, C.C. Huang, Y.C. Liang, Y.C. Tsai, K.S. Hsu, Impairment of long-term depression in the anterior cingulate cortex of mice with bone cancer pain, Pain 153 (2012) 2097–2108. [4] K.R. Gong, F.L. Cao, Y. He, C.Y. Gao, D.D. Wang, H. Li, F.K. Zhang, Y.Y. An, Q. Lin, J. Chen, Enhanced excitatory and reduced inhibitory synaptic transmission contribute to persistent pain-induced neuronal hyper-responsiveness in anterior cingulate cortex, Neuroscience 171 (2010) 1314–1325. [5] M. Han, X. Xiao, Y. Yang, R.Y. Huang, H. Cao, Z.Q. Zhao, Y.Q. Zhang, SIP30 is required for neuropathic pain-evoked aversion in rats, Journal of Neuroscience: Official Journal of the Society for Neuroscience 34 (2014) 346–355. [6] K. Hargreaves, R. Dubner, F. Brown, C. Flores, J. Joris, A new and sensitive method for measuring thermal nociception in cutaneous hyperalgesia, Pain 32 (1988) 77–88. [7] T. Hirai, K. Chida, Protein kinase Czeta (PKCzeta): activation mechanisms and cellular functions, Journal of Biochemistry 133 (2003) 1–7. [8] J.P. Johansen, H.L. Fields, Glutamatergic activation of anterior cingulate cortex produces an aversive teaching signal, Nature Neuroscience 7 (2004) 398–403. [9] J.P. Johansen, H.L. Fields, B.H. Manning, The affective component of pain in rodents: direct evidence for a contribution of the anterior cingulate cortex, Proceedings of the National Academy of Sciences of the United States of America 98 (2001) 8077–8082. [10] M.T. Kelly, J.F. Crary, T.C. Sacktor, Regulation of protein kinase Mzeta synthesis by multiple kinases in long-term potentiation, Journal of Neuroscience: Official Journal of the Society for Neuroscience 27 (2007) 3439–3444. [11] T. King, C. Qu, A. Okun, O.K. Melemedjian, E.K. Mandell, I.Y. Maskaykina, E. Navratilova, G.O. Dussor, S. Ghosh, T.J. Price, F. Porreca, Contribution of
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Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Upregulation of glutamatergic transmission in anterior cingulate cortex in the diabetic rats with neuropathic pain Weifang Li a , Peng Wang b , Hua Li a,∗ a b
Department of Geriatric Endocrinology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan 450001, China Department of Epidemiology, College of Public Health, Zhengzhou University, Zhengzhou, Henan 450001, China
h i g h l i g h t s • STZ induced hyperglycemia, thermal hyperalgesia and tactile allodynia in rats. • Enhanced glutamatergic transmission in the ACC neurons of the modeled rats. • Increased PKM phosphorylation in ACC of the modeled rats.
a r t i c l e
i n f o
Article history: Received 26 January 2014 Received in revised form 21 February 2014 Accepted 7 March 2014 Keywords: Diabetic neuropathic pain Anterior cingulate cortex Glutamatergic transmission Protein kinase M
a b s t r a c t Peripheral neuropathic pain is a common complication in the diabetic patients, and the underlying central mechanism remains unclear. Forebrain anterior cingulate cortex (ACC) is critically involved in the supraspinal perception of physical and affective components of noxious stimulus and pain modulation. Excitatory glutamatergic transmission in the ACC extensively contributed to the maintenance of negative affective component of chronic pain. The present study examined the adaptation of glutamatergic transmission in the ACC in rats with diabetic neuropathic pain. Injection with streptozotocin (STZ) induced hyperglycemia, thermal hyperalgesia and mechanical allodynia in the rats. In these rats, significant enhanced basal glutamatergic transmission was observed in the ACC neurons. The increased presynaptic glutamate release and enhanced conductance of postsynaptic glutamate receptors were also observed in the ACC neurons of these modeled rats. Increased phosphorylation of PKM, but not the expression of total PKM, was also observed in the ACC. Microinjection of PKM inhibitor ZIP into ACC attenuated the upregulation of glutamate transmission and painful behaviors in STZ-injected rats. These results revealed a substantial central sensitization in the ACC neurons in the rodents with diabetic neuropathic pain, which may partially underlie the negative affective components of patients with diabetic neuropathic pain. © 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Peripheral neuropathic pain is a common complication in the diabetic patients. While a number of studies demonstrate that significant plasticity occurs in the spinal nocisponsive neurons, emerging evidences indicate significant adaptation in supraspinal brain regions pertinent to the processing of pain information. Forebrain anterior cingulate cortex (ACC), a brain region processing the cognitive and emotional functions of the brain, is critically involved in the supraspinal perception of noxious stimulus and pain modulation [27]. Previous anatomical, behavioral and physiological evidences suggest that neuronal circuit within and among ACC and
∗ Corresponding author. Tel.: +86 371 66913114. E-mail address: [email protected] (H. Li). http://dx.doi.org/10.1016/j.neulet.2014.03.038 0304-3940/© 2014 Elsevier Ireland Ltd. All rights reserved.
other brain regions (e.g., amygdala) processes information relating to the affective and/or physical components of peripheral painful sensation [8,13]. Peripheral noxious stimuli significantly increased the excitability of anterior cingulate cortex neurons [19], and local lesions of the anterior cingulate cortex reduced acute nociceptive responses and injury-related aversive behaviors in rodent model of chronic pain [9,28]. It was also documented that significant upregulation of excitatory glutamatergic transmission was observed in the ACC neurons, which critically contributing to the transition from acute nociceptive sensation to persisted pain [27,28] as well as the perception of negative components of pain. While increasing evidences demonstrate the significant adaptation of peripheral sensory neurons and dorsal horn neurons in the diabetic neuropathic pain, the character of central sensitization in nocisponsive supraspinal brain regions, such as ACC, remain unexplored.
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Accumulating evidences demonstrate the significant adaptation of plasticity of central neurocircuits, either excitatory or inhibitory, occurs in the physiological, such as learning and memory, and pathological conditions such as drug abuse [20] and chronic pain [27]. Among these, enhanced glutamatergic transmission in spinal dorsal horn and supraspinal regions has been extensively recognized for its critical role in the transition of from acute to chronic pain [16]. Peripheral inflammation and nerve injury induce the increase of presynaptic glutamate release [25] and membrane trafficking of AMPA receptor subunit GluR1 [2] in the ACC neurons. Currently, it remains unclear whether significant adaptation of glutamatergic transmission occurs in the ACC neurons in the setting of diabetic neuropathic pain. Activation of several intracellular pathways substantially modulates the synaptic plasticity in the central neurons in the setting of chronic pain [28]. Recent studies showed that an atypical PKC, PKM, critically contributed to sensitization of glutamatergic transmission in spinal and supraspinal nocisponsive neurons in the condition of peripheral nerve injury [11,13]. In the present study, we also study the functional change of PKM in the ACC of the rodent model of diabetic neuropathic pain. 2. Materials and methods 2.1. Animal model of streptozotocin-induced diabetic rats Male Sprague-Dawley rats (180–220 g) were purchased from the institutional center of experimental animals. All experimental protocols were approved by the Institutional Animal Care and Use Committee, and were conducted in accordance with National Institutes of Health guidelines.
Diabetes was induced by a single intraperitoneal injection of streptozotocin (STZ; 60 mg/kg; Sigma–Aldrich) freshly dissolved in 0.9% sterile saline as previously described [12]. Blood glucose levels were assayed using ACCU-CHEK test strips (Roche Diagnostics, Indianapolis) at 2 and 6 weeks after STZ administration, and the weights were measured weekly. Age-matched rats injected with vehicle were used as controls. 2.2. Cannula implantation and behavioral test A 30 gauge stainless steel cannula with a 33 gauge stylet plug was bilaterally implanted 0.5 mm above the ACC [1]. The rats were allowed one week recovery prior to further study. CNQX (10 nmol/0.5 l per side), ZIP (10 nmol/0.5 l per side), or vehicle was daily injected into the ACC 3 days prior to the behavioral test at week 6. Mechanical allodynia and thermal hyperalgesia were assessed in rats to evaluate painful behavior. Mechanical sensitivity was assessed by using a series of von Frey filaments with logarithmic incremental stiffness (Stoelting Co., Wood Dale, IL), and 50% probability paw withdrawal thresholds were calculated with the up–down method [14]. The thermal nociceptive thresholds were measured using radiant heat [6], and the time for the rat to remove the paw from the thermal stimulus was electronically recorded as the paw withdrawal latency (PWL). 2.3. Whole-cell patch clamp recordings in ACC slices The electrophysiological recording was performed in the rats with behavioral tests. The coronal brain slices (300 m) containing
Fig. 1. STZ induced hyperglycemia, thermal hyperalgesia and mechanical allodynia in the rats. (A) Significantly increased blood glucose levels were observed in the rats at 2 and 6 weeks after STZ injection (n = 19–22 rats in each group). (B) Significant lower body weight was observed in the STZ-injected rats (n = 19–22 rats in each group). Time course of hindpaw withdrawal latency response to radiant thermal stimulation (C) and paw withdrawal threshold response to mechanical stimulation (D) in the rats treated with STZ or vehicle (n = 11 rats in each group). * P < 0.05, ** P < 0.01; *** P < 0.001.
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the ACC were obtained, and the whole-cell patch clamp recordings was performed on the ACC slices after 1-h recovery as previously reported [3]. Excitatory postsynaptic currents (EPSCs) were recorded from layer II/III neurons with a electrical stimulation (0.25 ms, 0.05–0.3 mA) in layer V in ACC. The membrane potential was held at −70 mV throughout the experiment. The glutamate synaptic strength was investigated with three graded stimulus intensities, and the input (stimulus intensity)-out (EPSC amplitude) response was plotted accordingly. For paradigm of paired-pulse ratios (PPR), a pair of EPSCs was evoked by two stimuli with an interval of 40, 60 and 80 ms. Four PPRs were averaged to obtain a mean PPR to minimum the variances among consecutive events. 2.4. Protein extraction and immunoblotting The rostral ACC tissues were punched from stereotaxically identified 2 mm thick slices (bregma +3.7 to +1.7) from the rats and proceeded to immunoblotting as described previously [11]. Primary antibodies against phospho (Thr 410) – PKM (Cell Signaling Technology, 1:1000), PKM (Cell Signaling Technology, 1:1000), -actin (Santa Cruz Biotechnology, 1: 1000) and appropriate secondary antibodies were used. The immunoreactivity was detected using enhanced chemiluminescence (ECL Advance Kit; Amersham Biosciences). The immunoreactivity of target proteins was normalized to that of -actin. 2.5. Drug and data analysis 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX), 2-amino-5phos-phonopentanoic acid (AP-5), ␣-amino-3-hydroxy-5-methyl4-isoxazolepropionic acid (AMPA), bicuculline and all other drugs were purchased from Sigma–Aldrich or Tocris, and were applied through the perfusion solution. Results were analyzed by paired or unpaired t-test, or two-way ANOVA followed by post hoc test. All data are expressed as mean ± S.E.M. P < 0.05 was considered statistically significant. 3. Results 3.1. Blood glucose levels and body weights of the experimental groups As shown in Fig. 1, the rats treated with STZ consistently showed significantly higher blood glucose levels (429 ± 27 mg/dl, n = 22 rats) compared to the rats treated with vehicle (142 ± 15 mg/dl, n = 19 rats) at 2 weeks after STZ injection (Fig. 1A). Such hyperglycemia was observed in all of the rats at 6 weeks after STZ injection (Fig. 1A). Meanwhile, the body weights of STZ-treated rats were significantly lower than those of vehicle-treated rats during 6 weeks of observation (Fig. 1B). These observations indicated that all of the rats that received STZ became diabetic, in accordance with previous reports [12]. 3.2. Significant thermal hyperalgesia and mechanical allodynia in the STZ-treated rats Consistent with previous reports [12,21], the STZ-injected rats showed significantly decreased hindpaw withdrawal latencies response to the radiant thermal stimuli (Fig. 1C), which obviously manifested at 2 weeks after the STZ injection, and maintained through at least 6 weeks after STZ injection. Similarly, significantly lower paw withdrawal thresholds response to mechanical stimuli were also observed from 2 weeks through at least 6 weeks after injection with STZ (Fig. 1D). These results confirmed the thermal hyperalgesia and mechanical allodynia in the rats injected with STZ.
Fig. 2. STZ increased glutamatergic strength in the ACC neurons. Significantly enhanced input (stimuli intensity)–output (EPSC amplitude) was observed in the ACC neurons of STZ-treated rats, which was attenuated by the microinjection of ZIP, a selective PKM inhibitor, into the ACC. n = 9–11 neurons per group. * P < 0.05; ** P < 0.01.
3.3. Significantly enhanced glutamatergic transmission in the ACC in the STZ-treated rats We then examined whether significant adaptation of glutamatergic transmission occurred in the ACC. The EPSCs were recorded in the presence of bicuculline (30 M) by the electric stimuli with three graded intensities. The evoked EPSCs were almost completely abolished by the application of AP-V (10 M) and CNQX (10 M) (146.2 ± 8.7 pA vs. 9.7 ± 3.6 pA, n = 9 neurons, P < 0.01), confirming the glutamatergic components. As shown in Fig. 2, significant left shift of the input–output response of the evoked EPSCs was observed in ACC neurons in the STZ-treated rats. Meanwhile, microinjection of CNQX (10 nmol/side) significantly attenuated the thermal hyperalgesia and mechanical allodynia in the rats injected with STZ (Fig. 3C and D). These suggested the enhanced glutamatergic strength in the ACC neurons of STZ-treated rats with neuropathic pain. 3.4. Significantly enhanced presynaptic glutamate release in the ACC in the STZ-treated rats Upregulation of glutamatergic transmission in the central neurons may result from the enhanced presynaptic glutamate release and/or the redistribution of the postsynaptic glutamate receptor subunits. Firstly, the paradigm of paired-pulse ratio (PPR) was applied to determine whether enhanced presynaptic glutamate release existed in the ACC neurons in the STZ-treated rats. As shown in Fig. 3A, the ACC neurons in STZ-injected rats had a PPR (inter-stimulation interval of 40 ms) of 1.19 ± 0.07 (n = 14 neurons), which was significantly less than that in the vehicle-treated rats (1.44 ± 0.08, n = 12 neurons, P < 0.05). The decreased PPR was consistently found at the stimuli with three different intervals (40 ms,
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Fig. 3. STZ increased presynaptic glutamate release and conductance of postsynaptic AMPA receptors in the ACC neurons. Significantly decreased PPR of evoked EPSCs was observed in the ACC neurons of STZ-treated rats at three different inter-stimuli intervals (A, n = 12 and 14 neurons in each group). Exogenous AMPA (1 M) elicited significantly stronger inward currents in the ACC neurons of the STZ-treated rats (B, n = 14 and 17 neurons in each group). Microinjection of CNQX (10 nmol/side) into the ACC significantly attenuated the thermal hyperalgesia and mechanical allodynia in the rats injected with STZ (C and D, n = 8–9 rats per group). Vehicle vs. STZ: * P < 0.05; ** P < 0.01; STZ vs. STZ + CNQX: ## P < 0.01.
60 ms, and 80 ms). These results suggested an enhanced presynaptic glutamate release in the ACC neurons in the STZ-treated rats. 3.5. Significantly enhanced inward current evoked by AMPA To determine whether adaptation of postsynaptic glutamate receptor exists in the ACC neurons in STZ-treated rats, exogenous AMPA (1 M) was perfused, and the inward current was recorded. As shown in Fig. 3B, AMPA (1 M) induced a significant larger inward current in the ACC neurons of the STZ-treated rats (135.4 ± 10.3 pA, n = 17 neurons, P < 0.01) than that in the vehicle-treated rats (89.6 ± 7.5 pA, n = 14 neurons). This suggested an adaptation of postsynaptic glutamate receptor in ACC neurons of the STZ-treated rats.
As shown in Fig. 4A, while the expression of total PKM remained unchanged, the phosphorylation of PKM (Thr 410) was significantly increased in the ACC tissues in the STZ-treated rats (n = 7–8 rats in each group). Bilateral microinjections of ZIP (10 nmol/side for 3 days), a selective cell-permeable PKM inhibitor, into the ACC significantly attenuated the upregulation of glutamatergic transmission (Fig. 2), and reduced the thermal hyperalgesia and mechanical allodynia in the rats injected with STZ (Fig. 4B and C). This implied, considering the functional significance of PKM in synaptic plasticity, that an upregulation of PKM signaling may account for the enhanced glutamatergic transmission in the ACC neurons of STZ-treated rats. 4. Discussion
3.6. Significantly increased expression of PKM It was well documented that upregulation of PKM signaling significantly contributed to the maintenance of synaptic plasticity in the brain neurons, and was critically involved in the central sensitization in several models of chronic pain [11,13]. Here immunoblotting technique was applied to detect the expression of PKM and its phosphorylation in the ACC tissues of the diabetic rats.
In the present study, we identified the enhanced glutamatergic transmission, via increased presynaptic glutamate release and enhanced conductance of postsynaptic AMPA receptor, in the ACC neurons in the diabetic neurons. We also found an increased phosphorylation of PKM, an atypical PKC, in the ACC, which possibly accounted for the enhanced glutamatergic transmission in these modeled rats. To our knowledge, this is the first investigation to
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Fig. 4. Significantly increased phosphorylation of PKM (Thr 410) was observed in the ACC tissues in the STZ-treated rats, while the expression of total PKM remained unchanged (n = 7 and 8 rats in each group). Microinjection of ZIP (10 nmol/side) into the ACC significantly attenuated the thermal hyperalgesia and mechanical allodynia in the rats injected with STZ (B and C, n = 8–9 rats per group). Vehicle vs. STZ: ** P < 0.01; STZ vs. STZ + ZIP: ## P < 0.01.
elucidate the central sensitization and the possible involvement of PKM in the in the anterior cingulate cortex, the key brain region for pain and emotion modulation, in the diabetic rats with neuropathic pain. Currently, a number of evidences demonstrated that the forebrain anterior cingulate cortex, a brain region processing the cognitive and emotional function, was critically involved in the perception of physical [27,28] and affective [5,13] components of pain. Significant depolarized membrane potential, lower firing threshold, lower input resistance and increased spike frequency were previously observed in the ACC of the rats with peripheral persistent painful stimuli [4]. Spinal injury induced the loss of the gray matter volume [23], and continuous inhibition of painful stimuli was associated with the increased gray matter in ACC in the patients with chronic pain [17]. In the present study, an enhanced glutamatergic transmission was, for the first time, revealed in the ACC neurons of the rats with diabetic neuropathic pain, which was attributable to the enhanced presynaptic glutamate release, indicated by decreased paired-pulse ratio of evoked EPSCs, and increased conductance of postsynaptic glutamate receptors, indicated by increased inward current evoked by exogenous AMPA (1 M). Previous studies ever found the similar enhanced glutamatergic transmission, along with the reduced inhibitory transmission, was observed in the ACC neurons in the rats with persistent painful stimuli [4], and suppression of the glutamatergic transmission in ACC neurons significantly attenuated the pain-evoked place escape/avoidance in the rat with chronic nerve constriction injury [5]. Significant increased presynaptic glutamate release and postsynaptic AMPA receptor subunit GluR1 membrane trafficking were ever reported in the ACC neurons of the
rats with chronic pain induced by peripheral inflammation [26] or nerve injury [22,25]. Similar adaptation of glutamatergic transmission in ACC was also observed in the rodent model with visceral pain [24], or bone-cancer pain [3]. Hence, it is postulated that similar redistribution of receptor subunits likely account for the increased conductance of postsynaptic AMPA receptors in the ACC of the diabetic rats. Taken together, these findings suggested that enhanced glutamate transmission in ACC neurons was a fundamental longterm plasticity during chronic pain, which would be responsible for the affective response to chronic pain and the aversion to noxious stimulus. PKM is an atypical PKC, which does not respond to either intracellular Ca2+ or diacylglycerol but is regulated by protein–protein interactions and potentially membrane lipid composition [7]. The expression of PKM is primarily restricted to neurons [15]. The character of autonomous activity suggests that PKM is an important player in LTP maintenance. Actually, strong synaptic stimulation, while inducing LTP, is associated with phosphorylation of PKM [10], and inhibition of PKM activity by a pseudosubstrate inhibitor, ZIP, significantly abolished the maintenance of LTP in the cortex [18]. Furthermore, peripheral nerve injury induced an early increase in PKM expression and a persistent increase in PKM phosphorylation in the ACC in the mice, and suppression of PKM activity reduced the upregulation of glutamatergic transmission in ACC neurons, as well as the mechanical allodynia, in the mice with neuropathic pain [13]. Here, a significant increased phosphorylation of PKM, but not the expression of total PKM, was observed in the ACC tissues, and inhibition of PKM activity alleviated the upregulation of glutamate transmission and pain behaviors in the rats with diabetic neuropathic pain. These results suggested that
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