- Open Access
Enhanced quantal release of excitatory transmitter in anterior cingulate cortex of adult mice with chronic pain
© Toyoda et al; licensee BioMed Central Ltd. 2009
- Received: 15 December 2008
- Accepted: 27 January 2009
- Published: 27 January 2009
The anterior cingulate cortex (ACC) is a forebrain structure that plays important roles in emotion, learning, memory and persistent pain. Our previous studies have demonstrated that the enhancement of excitatory synaptic transmission was induced by peripheral inflammation and nerve injury in ACC synapses. However, little information is available on their presynaptic mechanisms, since the source of the enhanced synaptic transmission could include the enhanced probability of neurotransmitter release at existing release sites and/or increases in the number of available vesicles. The present study aims to perform quantal analysis of excitatory synapses in the ACC with chronic pain to examine the source of these increases. The quantal analysis revealed that both probability of transmitter release and number of available vesicles were increased in a mouse model of peripheral inflammation, whereas only probability of transmitter release but not number of available vesicles was enhanced in a mouse model of neuropathic pain. In addition, we compared the miniature excitatory postsynaptic potentials (mEPSCs) in ACC synapses with those in other pain-related brain areas such as the amygdala and spinal cord. Interestingly, the rate and amplitude of mEPSCs in ACC synapses were significantly lower than those in the amygdala and spinal cord. Our studies provide strong evidences that chronic inflammatory pain increases both probability of transmitter release and number of available vesicles, whereas neuropathic pain increases only probability of transmitter release in the ACC synapses.
- Neuropathic Pain
- Anterior Cingulate Cortex
- Mechanical Allodynia
- Inflammatory Pain
- Transmitter Release
The ACC is involved in major brain functions including learning, memory, and persist pain [1–9]. Recently, a number of studies consistently suggest that the ACC plays important roles in processing pain-related information in humans and in the behavioral responses to noxious stimuli or tissue injury in animals [3, 6, 10–14]. As for the mechanisms of pain transmission and modulation, it has been proposed that the enhancement of synaptic transmission contributes to chronic pain. For example, we have shown that excitatory synaptic transmission was enhanced in the ACC of mice with persistent inflammatory pain  and neuropathic pain . There are at least two main evidences supporting for presynaptic changes of glutamate release in the ACC after the nerve injury: First, we demonstrated that paired-pulse facilitation (PPF), a phenomenon in which activation of a synapse at shorter intervals results in a presynaptic facilitation of transmitter release in response to the second stimulus  was apparently reduced in ACC synapses with chronic pain [7, 15]. Second, we showed that the rate of MK-801 (NMDA receptor antagonist) blocking was significantly faster in ACC synapses with chronic pain together with the increase of mEPSCs rate [7, 15]. Therefore, it is likely that peripheral inflammation or nerve injury produces increases in mean quantal content in ACC synapses.
The quantal content of transmission at a synapse is determined by the number of releasable quanta (N) that corresponds to the number of functional vesicles, and the probability (p) of each quantum to be released. Although our previous studies demonstrated that presynaptic neurotransmitter release was enhanced by peripheral inflammation and neuropathic pain in ACC synapses [7, 15], the exact changes in these quantal parameters remains to be investigated. To examine the source of the enhanced presynaptic neurotransmitter release, here we performed quantal analysis of excitatory synaptic transmission with inflammatory pain and neuropathic pain.
mEPSCs that are observed in the absence of presynaptic action potentials, are frequently used as a parameter to reflect altered synaptic transmission responsible for inflammatory pain , neuropathic pain  and diabetic neuropathy . The amplitude and decay time constant of mEPSCs are determined by various factors such as the glutamate concentration in the synaptic cleft and the receptor properties; the glutamate concentration in the synaptic cleft is determined by the kinetics of glutamate release, diffusion, binding by the receptors, uptake by the transporters, and synaptic geometry, while the receptor properties are determined by kinetics, density, and spatial distribution . We also compared the characteristics of mEPSCs in pain-related brain regions such as ACC, basolateral amygdala, and spinal cord of adult mice and discuss how such characteristics of mEPSCs maybe related to pain transmission and modulation.
Quantal analysis of excitatory synaptic transmission in ACC synapses following CFA injection and nerve ligation
Comparison of mEPSCs in ACC, basolateral amygdala, and spinal cord
We also compared the amplitude of mEPSCs among three groups. The amplitude of mEPSCs in ACC was significantly smaller than those in basolateral amygdala and spinal cord (p < 0.01 and p < 0.01, respectively), although there was no difference between basolateral amygdala and spinal cord (ACC, 9.8 ± 0.6 pA, n = 12; basolateral amygdala, 12.9 ± 0.4 pA, n = 14; spinal cord, 13.9 ± 1.4 pA, n = 12, Fig. 3C). Next, we compared the rise time of mEPSCs and found that there were no differences among three groups (ACC, 1.4 ± 0.1 ms, n = 12; basolateral amygdala, 1.4 ± 0.1 ms, n = 14; spinal cord, 1.2 ± 0.1 ms, n = 12, Fig. 3D). Finally, we compared the decay time of mEPSCs among three groups. The decay time of mEPSCs in ACC and basolateral amygdala were significantly larger than that in spinal cord (p < 0.01 and p < 0.01, respectively), although there was no difference between ACC and basolateral amygdala (ACC, 8.8 ± 0.8 ms, n = 12; basolateral amygdala, 8.6 ± 0.6 ms, n = 14; spinal cord, 5.7 ± 0.4 ms, n = 12, Fig. 3E). These results suggest that the rate and amplitude of glutamatergic mEPSCs in ACC synapses are lower than other pain-related areas at resting condition.
In the present study, we performed quantal analysis of excitatory synaptic transmission after CFA injection and nerve ligation. This is the first study, to our knowledge, to perform quantal analysis in the animal model of nerve injury. We demonstrate that both the probability of transmitter release and number of available vesicles were increased in mice following CFA injection, while only the probability of transmitter release but not the number of available vesicles was increased in mice with nerve ligation. Thus, it is strongly suggested that presynaptic mechanisms for enhanced excitatory synaptic transmission within the ACC may not be the same as comparing the inflammatory pain model with neuropathic pain model, although both models showed the enhancement of the presynaptic neurotransmitter release [7, 15].
Our current study provided the evidences for enhanced transmitter release by quantal analysis. However, we have to keep in mind that synaptic failures observed in this study would be smaller than expected, since we used the stimulus intensity just above the level that gave mostly failures. In general, when focal extracellular stimulation is used to obtain uEPSCs, the stimulus intensity is adjusted to value that demonstrates an all or none behavior of the EPSCs. Therefore, future studies would be necessary to precisely evaluate the probability of transmitter release and number of available vesicles.
Quantal analysis in the ACC of mice with inflammatory pain and nerve ligation
In mice following CFA injection, the amplitude of eEPSCs and the rate of mEPSCs were potentiated, whereas that of mEPSCs remained unchanged, compared with the results obtained from control mice. As demonstrated by quantal analysis, these observations can be explained by the enhancement of the number of available vesicles together with the increase in the probability of transmitter release. On the other hand, the enhancement of the amplitude of eEPSCs and mEPSCs together with the increase in mEPSC rate were observed in mice with nerve ligation, in comparison with the results obtained from control mice. These observations would be explained by the enhancement of the probability of transmitter release without changing the number of available vesicles and an increase in postsynaptic responsiveness. Taken together, these results indicate that the mechanisms of the enhanced synaptic transmission vary depending on the nerve injury models. Furthermore, these results would partly reflect the locus of synaptic alterations after peripheral nerve injury. For instance, in mice with inflammatory pain, we have previously shown that the enhanced synaptic transmission results from the increased probability of presynaptic neurotransmitter release rather than a possible postsynaptic modification of functional AMPA receptors , although we cannot completely rule out the possible involvement of postsynaptic mechanisms due to the enhancement of NR2B-containing receptors . In mice with nerve ligation, the enhanced synaptic transmission was mediated by presynaptic enhancement of glutamate release probability as well as postsynaptic enhancement of AMPA receptor-mediated responses because of the phosphorylation of GluR1 subunit . Increases in mEPSC amplitude after nerve ligation could also reflect the increase in the transmitter content of individual vesicles, provided that the postsynaptic sites were not saturated. Since it has previously been shown that the infusion of a high concentration of L-glutamate into mature calyceal terminals did not cause saturation of AMPA receptors , enhancement of the transmitter content of individual vesicles may have been caused by nerve ligation. Taken together, our results thus provide the evidence that excitatory synaptic transmission is subject to divergent plasticity in different peripheral nerve injury models.
The strength of a synaptic connection between two neurons reflects three quantal parameters of neurotransmission: the number of a pool of available quanta (N); the probability of transmitter release (p); and the amplitude of the response attributable to quantal release (q) . Although we defined N as the number of available vesicles, it is generally believed that N corresponds to the number of release sites or active zones that contain clusters of vesicles, some of which are docked near the presynaptic membrane . Thus, it is likely that the enhanced quantal content with inflammatory pain reflects an increase in the probability of release of available quanta, with perhaps also increases in the number of release sites. In other words, two types of changes can be conceivable in ACC synapses with peripheral inflammation: (1) increase in the probability of activating exocytosis of a docked vesicle or (2) increase in the probability that a release site is occupied by a docked vesicle ready for release . It has been suggested that 'kiss-and-run' vesicle fusion exists at endocrine cells, pituitary nerve terminals, cultured hippocampal neurons, and calyx of Held synapses . The kiss-and-run can rapidly recycle vesicles and affect the size and the kinetics of the synaptic current. Although little is known whether kiss-and-run plays a major mode of fusion in ACC synapses, such mode of fusion may also be altered with inflammatory pain and nerve ligation. Future studies are necessary to understand the relationship between the enhanced quantal content and 'kiss-and-run' vesicle fusion at ACC synapses with chronic pain.
Calcium-stimulated adenylyl cyclase and possible presynaptic action
We have previously shown that adenylyl cyclase  1 and 8 are essential for chronic pain. For example, in AC1/8 double knockout mice, chronic pain sensitization was significantly reduced in inflammatory  and neuropathic pain model [7, 21]. In ACC synapses with inflammatory pain, inhibition of AC1 and/or AC8 were sufficient to inhibit the long-lasting enhanced presynaptic transmitter release . Thus, the molecular mechanisms responsible for the enhancement of probability of transmitter release and number of available vesicles in inflammatory pain could be mediated by presynaptic AC1 and/or AC8 proteins such as cAMP response element binding protein (CREB) . In addition, we have found that AC1 is critical for both long-term presynaptic and postsynaptic changes in ACC synapses after nerve ligation . AC1 thus may contribute both presynaptically and postsynaptically to nerve ligation-induced plastic changes in the ACC. Therefore, AC1 may serve as a potential therapeutic target for treating chronic pain.
Characteristics of mEPSCs in pain related brain regions
Results obtained from the analysis of mEPSCs reveal that the rate and the amplitude of mEPSCs in spinal cord are significantly lower than those in ACC and basolateral amygdala. Nociceptive transmission starts from the peripheral terminals of dorsal root ganglion (DRG) to DRG soma, then to spinal cord dorsal horn and finally reaches to supraspinal structures such as brainstem, thalamus and pain-related cortex including ACC and insular cortex. Considering the nociceptive transmission pathway, spinal cord plays essential roles as not only the first site of synaptic integration for pain transmission but also the site to convey pain transmission to the supraspinal structure. Therefore, the excitability of the spinal dorsal horn neurons may be maintained at a higher level than other pain-related brain areas even at resting condition. Why are the rate and amplitude of mEPSCs in ACC synapses significantly lower than those in basolateral amygdala and spinal cord? Although it remains unclear why ACC synapses display low-rate low-amplitude mEPSCs compared to basolateral amygdala and spinal cord, it is strongly suggested that glutamatergic input to ACC synapses is low at resting condition. Probably, the excitability of ACC synapses may be maintained at a lower level under the resting condition, because ACC has to be activated in case of placebo analgesia/opioid analgesia as well as pain perception, two opposite physiological processes . Thus, a systemic analysis of mEPSCs can provide us detailed information on synaptic transmission under physiological and pathological pain conditions. Future studies on quantal analysis in other pain-related brain regions with inflammatory and neuropathic pain will expand our knowledge to understand the synaptic mechanisms underlying chronic pain.
In summary, we demonstrate the strong evidences that the enhancement of both the probability of transmitter release and number of available vesicles contributes to the enhanced synaptic efficacy in ACC synapses with inflammatory pain, whereas only the enhancement of the probability of transmitter release underlies the enhanced excitatory synaptic transmission in ACC synapses with nerve ligation. Thus, peripheral nerve injury models are not all equivalent with respect to the excitatory synaptic transmission in ACC synapses. The mechanisms underlying these differences provide critical synaptic insights into physiological and pathological response to chronic pain and need to be clarified to alleviate chronic pain.
All mice were maintained on a 12 h light/dark cycle with food and water provided ad libitum. The Animal Care and Use Committee of the University of Toronto approved all experimental procedures. Adult (6–8 weeks) male mice were used in this study.
Inflammatory pain and neuropathic pain models
To induce inflammatory pain, 10 μl of 50% CFA (Sigma, St. Louis, MO) was injected subcutaneously into the dorsal surface of one hindpaw [14, 15]. As a sham group, saline was injected. A model of neuropathic pain was induced by the ligation of the common peroneal nerve (CPN) as described previously . Briefly, mice were anesthetized by intraperitoneal injection of a mixture saline of ketamine and xylazine. The left CPN was ligated with chromic gut suture 5-0 (Ethicon) slowly until contraction of the dorsiflexors of the foot was visible as twitching of the digits. The mechanical allodynia was tested on postsurgical day 7, and the mice were used for electrophysiological studies on postsurgical days 7–14. Sham surgery was done on the left leg by incising the skin, subcutaneous fascia, and the intercompartmental fascia. The muscle was then pulled laterally and posteriorly to expose the nerve. Thereafter, the skin was sutured with 5-0 silk suture.
For the measurement of behavioral responses, mice were placed in a round container and allowed to acclimate for 30 min before testing. Mechanical allodynia was assessed based on the responsiveness of the hindpaw to the application of von Frey filaments (Stoelting) to the point of bending. Positive responses include licking, biting, and sudden withdrawal of the hindpaw. Experiments were performed to characterize the threshold stimulus. Mechanical pressure from a 1.65 filament (force, 0.008 g) was found to be innocuous in normal mice. This filament was then used to test the mechanical allodynia after CFA injection and nerve ligation. Mechanical allodynia was tested five times with an intertrial interval of 10 min. Animals were then permitted a rest period for 20 min, after which mechanical allodynia was again tested. After obtaining the positive responses, the mice were used for electrophysiological recordings.
Coronal brain slices (300 μm) containing the ACC, basolateral amygdala, and spinal cord were prepared as described previously [15, 27, 28]. Slices were transferred to submerged recovery chamber with oxygenated (95% O2 and 5% CO2) artificial CSF containing the following (in mM): 124 NaCl, 2.5 KCl, 2 CaCl2, 2 MgSO4, 25 NaHCO3, 1 NaH2PO4, and 10 glucose at room temperature for at least 1 h.
Whole-cell patch-clamp recordings
Whole-cell patch-clamp recordings were performed in a recording chamber on the stage of an Axioskop 2FS microscope (Zeiss, Oberkochen, Germany) with infrared differential interference contrast optics for visualization of whole-cell patch-clamp recording. Evoked EPSCs (eEPSCs) were recorded from layer II/III neurons with an Axopatch 200 B amplifier (Molecular Devices, CA), and the stimulations were delivered by a bipolar tungsten stimulating electrode placed in layer V of the ACC. Unitary EPSCs (uEPSCs) were obtained by repetitive stimulations for 100 trials at 0.1 Hz, and neurons were voltage clamped at -70 mV. The stimulus threshold was determined by applying a series of stimuli with increasing intensities until detectable currents were obtained, and the stimulus intensity (5 V) just above the level that gave mostly failures was used in the present study. Average amplitude and coefficient of variation (CV) of uEPSCs were calculated from 100 consecutive sweeps. The recording pipettes (3–5 MΩ) were filled with solution containing the following (in mM): 145 K-gluconate, 5 NaCl, 1 MgCl2, 0.2 EGTA, 10 HEPES, 2 Mg-ATP, and 0.1 Na3-GTP, adjusted to pH 7.2 with KOH. For mEPSC recording in the ACC, 1 μM TTX was added in the perfusion solution following eEPSCs recordings. For mEPSC recording in the ACC, basolateral amygdala, and spinal cord, 1 μM TTX was added in the perfusion solution. Picrotoxin (100 μM) was always present to block GABAA receptor-mediated inhibitory synaptic currents in all experiments. Access resistance was 15–30 MΩ and monitored throughout the experiment. Data were discarded if access resistance changed >15% during an experiment. Results were expressed as mean ± SE except the amplitude of uEPSCs (mean ± SD). Rise time and decay time were defined as reported previously . Statistical comparisons were performed using t-test or ANOVA followed by Fisher's PLSD (protected least significant difference) post-hoc test. In all cases, p < 0.05 was considered statistically significant. Since no differences were found between saline-injected and sham operated groups, these data were pooled in a single control group.
We would like to thank Dr. Ling-Gang Wu for his valuable comment. This work is supported by grants from the Canadian Institutes of Health Research (CIHR81086 and 84256), the EJLB-CIHR Michael Smith Chair in Neurosciences and Mental Health, and the Canada Research Chair to Dr. Min Zhuo.
- Devinsky O, Morrell MJ, Vogt BA: Contributions of anterior cingulate cortex to behaviour. Brain 1995, 118: 279–306.PubMedView ArticleGoogle Scholar
- Zhuo M: A synaptic model for pain: long-term potentiation in the anterior cingulate cortex. Mol Cells 2007, 23: 259–271.PubMedGoogle Scholar
- Zhuo M: Molecular mechanisms of pain in the anterior cingulate cortex. J Neurosci Res 2006, 84: 927–933.PubMedView ArticleGoogle Scholar
- Zhuo M: Central plasticity in pathological pain. Novartis Found Symp 2004, 261: 132–145. discussion 145–154.PubMedView ArticleGoogle Scholar
- Frankland PW, Bontempi B, Talton LE, Kaczmarek L, Silva AJ: The involvement of the anterior cingulate cortex in remote contextual fear memory. Science 2004, 304: 881–883.PubMedView ArticleGoogle Scholar
- Wei F, Qiu CS, Liauw J, Robinson DA, Ho N, Chatila T, Zhuo M: Calcium calmodulin-dependent protein kinase IV is required for fear memory. Nat Neurosci 2002, 5: 573–579.PubMedView ArticleGoogle Scholar
- Xu H, Wu LJ, Wang H, Zhang X, Vadakkan KI, Kim SS, Steenland HW, Zhuo M: Presynaptic and postsynaptic amplifications of neuropathic pain in the anterior cingulate cortex. J Neurosci 2008, 28: 7445–7453.PubMed CentralPubMedView ArticleGoogle Scholar
- Zhao MG, Toyoda H, Lee YS, Wu LJ, Ko SW, Zhang XH, Jia Y, Shum F, Xu H, Li BM, et al.: Roles of NMDA NR2B subtype receptor in prefrontal long-term potentiation and contextual fear memory. Neuron 2005, 47: 859–872.PubMedView ArticleGoogle Scholar
- Zhuo M: Cortical excitation and chronic pain. Trends Neurosci 2008, 31: 199–207.PubMedView ArticleGoogle Scholar
- Davis KD, Pope GE, Crawley AP, Mikulis DJ: Neural correlates of prickle sensation: a percept-related fMRI study. Nat Neurosci 2002, 5: 1121–1122.PubMedView ArticleGoogle Scholar
- Rainville P, Duncan GH, Price DD, Carrier B, Bushnell MC: Pain affect encoded in human anterior cingulate but not somatosensory cortex. Science 1997, 277: 968–971.PubMedView ArticleGoogle Scholar
- Singer T, Seymour B, O'Doherty J, Kaube H, Dolan RJ, Frith CD: Empathy for pain involves the affective but not sensory components of pain. Science 2004, 303: 1157–1162.PubMedView ArticleGoogle Scholar
- Wager TD, Rilling JK, Smith EE, Sokolik A, Casey KL, Davidson RJ, Kosslyn SM, Rose RM, Cohen JD: Placebo-induced changes in FMRI in the anticipation and experience of pain. Science 2004, 303: 1162–1167.PubMedView ArticleGoogle Scholar
- Wu LJ, Toyoda H, Zhao MG, Lee YS, Tang J, Ko SW, Jia YH, Shum FW, Zerbinatti CV, Bu G, et al.: Upregulation of forebrain NMDA NR2B receptors contributes to behavioral sensitization after inflammation. J Neurosci 2005, 25: 11107–11116.PubMedView ArticleGoogle Scholar
- Zhao MG, Ko SW, Wu LJ, Toyoda H, Xu H, Quan J, Li J, Jia Y, Ren M, Xu ZC, Zhuo M: Enhanced presynaptic neurotransmitter release in the anterior cingulate cortex of mice with chronic pain. J Neurosci 2006, 26: 8923–8930.PubMedView ArticleGoogle Scholar
- Zucker RS, Regehr WG: Short-term synaptic plasticity. Annu Rev Physiol 2002, 64: 355–405.PubMedView ArticleGoogle Scholar
- Wang XL, Zhang HM, Chen SR, Pan HL: Altered synaptic input and GABAB receptor function in spinal superficial dorsal horn neurons in rats with diabetic neuropathy. J Physiol 2007, 579: 849–861.PubMed CentralPubMedView ArticleGoogle Scholar
- Glavinovic MI: Mechanisms shaping fast excitatory postsynaptic currents in the central nervous system. Neural Comput 2002, 14: 1–19.PubMedView ArticleGoogle Scholar
- Bekkers JM, Stevens CF: Presynaptic mechanism for long-term potentiation in the hippocampus. Nature 1990, 346: 724–729.PubMedView ArticleGoogle Scholar
- Zucker RS: Short-term synaptic plasticity. Annu Rev Neurosci 1989, 12: 13–31.PubMedView ArticleGoogle Scholar
- Wei F, Qiu CS, Kim SJ, Muglia L, Maas JW, Pineda VV, Xu HM, Chen ZF, Storm DR, Muglia LJ, Zhuo M: Genetic elimination of behavioral sensitization in mice lacking calmodulin-stimulated adenylyl cyclases. Neuron 2002, 36: 713–726.PubMedView ArticleGoogle Scholar
- Randic M: Plasticity of excitatory synaptic transmission in the spinal cord dorsal horn. Prog Brain Res 1996, 113: 463–506.PubMedView ArticleGoogle Scholar
- Yamashita T, Ishikawa T, Takahashi T: Developmental increase in vesicular glutamate content does not cause saturation of AMPA receptors at the calyx of held synapse. J Neurosci 2003, 23: 3633–3638.PubMedGoogle Scholar
- Zucker RS: Changes in the statistics of transmitter release during facilitation. J Physiol 1973, 229: 787–810.PubMed CentralPubMedView ArticleGoogle Scholar
- He L, Wu LG: The debate on the kiss-and-run fusion at synapses. Trends Neurosci 2007, 30: 447–455.PubMedView ArticleGoogle Scholar
- Vadakkan KI, Jia YH, Zhuo M: A behavioral model of neuropathic pain induced by ligation of the common peroneal nerve in mice. J Pain 2005, 6: 747–756.PubMedView ArticleGoogle Scholar
- Xu H, Wu LJ, Zhao MG, Toyoda H, Vadakkan KI, Jia Y, Pinaud R, Zhuo M: Presynaptic regulation of the inhibitory transmission by GluR5-containing kainate receptors in spinal substantia gelatinosa. Mol Pain 2006, 2: 29.PubMed CentralPubMedView ArticleGoogle Scholar
- Wu LJ, Ko SW, Toyoda H, Zhao MG, Xu H, Vadakkan KI, Ren M, Knifed E, Shum F, Quan J, et al.: Increased anxiety-like behavior and enhanced synaptic efficacy in the amygdala of GluR5 knockout mice. PLoS ONE 2007, 2: e167.PubMed CentralPubMedView ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.