Research Paper

Forebrain NMDA receptors contribute to neuronal spike responses in adult mice

WANG Guo-Du, ZHUO Min^*

Department of Physiology, University of Toronto Center for the Study of Pain, University of Toronto, 1 King´s College Circle, Toronto, Ontario M5S 1A8, Canada

Abstract: Glutamate is the major fast excitatory transmitter in the central nervous system. While normal synaptic transmission is mediated by a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainate receptors, N-methyl-D-aspartate (NMDA) receptors are thought to selectively contribute to plasticity. Genetically enhancing NMDA receptor functions enhances animal behavior in normal physiological learning and enhances their sensitivity in the case of tissue injury. One major mechanism for NMDA receptors is synaptic long-term potentiation (LTP). Here we present evidence that NMDA receptors not only contribute to normal synaptic responses induced by stimulation of local layer V or white matters, but also contribute to generation of action potentials induced by a depolarizing step applied to the soma. Calcium-calmodulin sensitive adenylyl cyclase 1 and cAMP signal pathways likely mediate these effects. Considering the importance of cingulate neurons in nociception and pain, our results provide a new mechanism for NMDA receptor contributing to neuronal synaptic transmission, spiking properties in forebrains, and possible forebrain-related behavioral nociceptive responses and pain.

Key words: anterior cingulate cortex; NMDA receptor; adenylyl cyclase; action potential; cAMP; mice

成年小鼠前脑NMDA受体参与神经元的动作电位发放

王过渡，卓 敏^*

多伦多大学医学院生理系，多伦多大学痛觉研究中心，多伦多，安大略省 M5S 1A8，加拿大

摘 要：谷氨酸是中枢神经系统主要的快速兴奋性递质。AMPA受体和海人藻酸受体主要参与突触传递，而NMDA受体 主要参与突触可塑性。基因操作的方法增强NMDA受体的功能，可以增强动物在正常生理状态下的学习能力，及在组织损 伤情况下的反应敏感性。NMDA受体参与生理功能的主要机制是长时程增强(long-term potentiation, LTP)。我们的研究表明，NMDA受体不仅参与刺激前扣带皮层的第五层细胞或刺激白质诱导的突触反应，而且参与在胞体施加去极化跃阶电流诱导的 动作电位的发放。钙-钙调蛋白敏感的腺苷酸环化酶1 (adenylyl cyclase 1, AC1)和cAMP信号通路可能介导了这些反应。由于扣带皮层神经元在伤害性刺激和痛中发挥重要作用，我们的结果为前脑NMDA受体参与突触传递和动作电位发放，以及与前 脑相关的行为，如感受伤害性刺激和痛，提供了一个新的机制。

关键词：前扣带皮层；NMDA受体；腺苷酸环化酶；动作电位；cAMP；小鼠

中图分类号：Q424；Q427

Forebrain neurons are important for pain-related perception^[1-3]. Recent studies from both human and animals consistently suggest that the anterior cingulate cortex (ACC) and its related areas are important for processing pain perception. Lesions of the medial frontal cortex, including the ACC, significantly inhibited acute nociceptive responses as well as injury-related aversive memory behaviors^[4,5]. In patients with frontal lobotomies or cingulotomies, the unpleasantness of pain is abolished^[6-8]. Electrophysiological recordings and anatomic studies from both animals and humans demonstrate that neurons within the ACC, including layer II, III and V neurons, respond to noxious stimuli, including nociceptive specific neurons^[9-16]. Neuroimaging studies further confirm these observations and show that the ACC, together with other cortical structures, are activated by acute noxious stimuli^[1,3,17-21]. Furthermore, recent studies in both anesthetized and freely moving animals show that stimulation of the ACC facilitated the spinal nociceptive tail-flick reflex^[22] and generated fear memory^[23]. Thus, understanding of synaptic mechanisms within the ACC will greatly help us gain insights into plastic changes in the brain related to central pain.

NMDA receptors are important for activity-synaptic plastic changes in the central nervous system^[24,25]. NMDA receptors, including NR1, NR2A and NR2B, are highly expressed in forebrain areas^[26,27]. Systemic application of NMDA receptor antagonists inhibited inflammation-related persistent pain in animals and chronic pain in humans with a recent note that NR2B receptor antagonists may have less side effects^[8,28]. Recent studies using genetically manipulated mice generated further evidence for the roles of NMDA receptors in persistent pain. Genetic deletion of PSD93 significantly reduced NMDA receptor-mediated currents in the spinal dorsal horn neurons/ACC neurons and persistent pain^[29]. In transgenic mice with forebrain-targeted NR2B overexpression^[30,331], inflammation-related pain was selectively enhanced^[27]. Local microinjection of the NMDA receptor antagonist AP-5 into the ACC blocked inflammation-related allodynia^[15].

One possible function of NMDA receptors in the ACC is that its activation contributes to the enhancement of excitatory synaptic transmission in the ACC neurons^[32]. Indeed, excitatory synaptic transmission in the ACC is mainly mediated by AMPA receptors, and activation of NMDA receptors is required for the enhancement of synaptic transmission in the ACC. Our recent studies in the adult mouse ACC slices suggested that NMDA receptors can also contribute to normal synaptic transmission, in particular at temperatures close to physiological conditions^[33]. These slow responses, mediated by NMDA receptors can also be recorded in both anesthetized and freely moving mice, suggesting that NMDA receptors likely conduct normal synaptic transmission in the ACC. Because several downstream signaling pathways can be activated by NMDA receptors, here we would like to explore if tonic activation of NMDA receptors in the ACC may contribute to the generation of action potentials in the ACC neurons. Using wild type (WT)and gene knockout mice lacking adenylyl cyclase subtype 1(AC1), we showed evidence that in the adult mouse (C57/6) ACC NMDA receptors can be functional in normal conditions.

1 MATERIALS AND METHODS

1.1 Animals

Adult male 12~16 week-old mice (C57/6J; Jackson laboratory) were used. AC1 knockout mice and littermate WT mice were provided by Dr. Louis Muglia in Washington University. To most closely match mice for background yet efficiently generate the large number of mice used in these studies, we generated WT/WT, AC1 KO/AC8 WT, AC8 KO/AC1 WT, and AC1 KO/AC8 KO breeders from AC1 het/AC8 het matings, and used the offspring from these breeders for the described studies. To minimize drift of background in a given genotype line, we used several breeding pairs. Both WT and mutant mice were well groomed and showed no signs of abnormality or any obvious motor defects. As it was impossible to visually distinguish mutant mice from WT mice, experimenters were blind to genotype. The experimental protocol was approved by the Animal Studies Committee at Washington University and University of Toronto.

1.2 Slice preparation

Mice were anaesthetized with urethane as previously described^[34]. Transverse brain slices, 450~500 mm, were obtained from male adult mice. Slices were kept in a plastic chamber containing oxygenated saline (in mmol/L: NaCl 124; KCl 4; NaHCO₃ 26; NaH₂PO₃ 2; CaCl₂ 2; MgSO₄ 1; D-glucose 10) at 32 ℃ for at least 2~3 h.

1.3 Electrophysiology

After a slice was transferred to the recording chamber, a bipolar tungsten stimulating electrode was placed in the white matter or adjacent to layer V. Intracellular recordings of synaptic responses were performed from neurons located in the layer V with a 4 mol/L potassium acetate-filled glass microelectrode (75~200 MW). Postsynaptic excitatoty postsynaptic potentials (EPSPs) were evoked at 0.01~ 0.02 Hz. As reported previously^[35], neurons in the cingulated layer V received high density of innervations from the thalamus. Monosynaptic EPSPs were identified using two criteria: (1) the response latency did not change with increasing intensities of electrical stimulation; and (2) repetitive stimulation did not change response latency. EPSPs of the neurons were evoked with electrical stimulation at different intensities (0.2 ms, 1~30 V) and recorded through a high-input impedance bridge circuit of amplifier (Axonclamp 2B, Axon Instruments) and stored in pClamp (Axon Instruments) files. The perfusion artificial cerebral spinal fluid (ACSF) was oxygenated with 95% O₂ and 5% CO₂. The temperature and perfusion rate of recording were kept at 34 ℃ and 2~5 ml/min, respectively.

1.4 Pharmacology

All drugs were purchased from RBI-Sigma. In all experiments, bicuculline methiodide (10 μmol/L) was added to the perfusion solution to block inhibitory transmission.

1.5 Data analyses

Data are presented as means±SEM. Statistical comparisons were made with the use of one-way analyses of variance (ANOVAs) or Student's t-test. P<0.05 was considered significant.

2 RESULTS

2.1 NMDA receptors contribute to spike generation in ACC pyramidal cells

We have performed intracellular recordings from pyramidal cells of ACC slices of adult mice. To determine if tonic NMDA receptors may contribute to neuronal spike firing due to the postsynaptic depolarization, we used bath application of selective NMDA receptor antagonist AP-5. Bath application of AP-5 (100 mmol/L) significantly reduced action potentials induced by postsynaptic injection of currents (0.6~0.8 nA) (n=17 cells). The inhibitory effect of AP-5 was relatively fast and typically occurred within 1~3 min after the start of AP-5 perfusion. The spike responses recovered after the washout of AP-5, indicating that the effect is reversible (Fig.1).

To test if the spikes are sensitive to the blockade of sodium channels, we perfused a selective sodium channel blocker TTX (1 μmol/L) to confirm that these spike responses can be blocked. As expected, bath application of TTX completely blocked all spike responses in cingulate pyramidal cells (n=12 cells; Fig.2).

2.2 Synaptically activated NMDA receptors

Next, we placed a stimulating electrode in the ACC to detect if synaptically activated NMDA receptor may also contribute to spike generation in cingulate neurons. As shown in Fig.3, local electrical stimulation induced EPSPs as well as action potentials. The generations of spikes are clearly intensity-dependent. Stimulation at low intensities usually only induced EPSPs. Previous studies in cingulate slices and freely moving animals consistently showed that postsynaptic NMDA receptors in cingulate neurons contribute to synaptic transmission^[23,33]. We thus decided to examine if inhibiting NMDA receptors may also affect synaptically activated spike responses. As shown in Fig.3, bath application of AP-5 significantly reduced synaptically induced spike responses (n=8). The inhibitory effect of AP-5 is reversible and recovered after the washout of AP-5. We can not completely rule out the possibility that AP-5 inhibitory effect may be in part due to reduced synaptic transmission.

2.3 Effects of soma hyperpolarization

It is well documented that the magnesium blockade of NMDA receptors is voltage-dependent. The contribution of NMDA receptors to spike generation may be due to NMDA receptors located at distal dendritic areas, since the resting potentials of these neurons are around ?0 mV (n=46). To test this possibility, we injected negative currents into the soma (to ?44 mV) and then applied local electrical stimulation (Fig.4A). If the NMDA receptors are located near the soma, we expect that AP-5 should induce less or no effect on spike responses. However, as shown in Fig.4, we found that AP-5-sensitive spike still persisted (n=6 cells). These findings suggest that NMDA receptors that contribute to spike generation are likely located distally to the soma. We did notice that AP-5 also affected EPSPs in some of the recordings (Fig.4A). Thus, we cannot completely rule out that some of these NMDA receptors may contribute to spike generation by increasing EPSPs (Fig.4)

2.4 Activation of adenylyl cyclases

Among many signaling proteins which act downstream from NMDA receptors, calcium-activated adenylyl cyclases are reported to be important for NMDA receptor-related functions^[32,36,37]. In the ACC neurons, both forms of calcium-activated AC1 and AC8 are highly expressed^[38]. Therefore, we wanted to examine if bath application of adenylyl cyclase activator forskolin may increase spike responses in the ACC neurons. Indeed, bath application of forskolin at a dose of 10 μmol/L was found to significantly enhance synaptic responses in the ACC neurons (n=24 cells; Fig.5). We also performed experiments with local electrical stimulation in the ACC slices. Application of forskolin produced significant enhancement of spike responses as well as synaptic EPSPs (n=12 cells; Fig.6).

2.5 Calcium-activated AC1

AC1 is the major calcium-activated adenylyl cyclase in the central neurons and it is most sensitive to calcium increase^[36]. To determine if AC1 may contribute to the enhancement of spike responses, we performed recordings from cingulate neurons of AC1 knockout mice. We found that forskolin failed to induce any more increases of spike responses (n=8 cells/5 mice; Fig.7A). By contrast, forskolin application induced increases in spike responses in WT littermate mice (n=4 cells/3 mice).

In Fig.7B, we summarized the present results in a simplified model. Tonic activation of NMDA receptors in the ACC neurons led to activation of calcium-stimulated AC1 in these neurons. Activation of AC1 led to increases of cAMP levels. The increased level of cAMP acted through unknown mechanism to recruit more action potentials.

3 DISCUSSION

The present studies provide strong evidence for the involvement of the NMDA receptors in the generation of action potentials in adult cingulate neurons. In addition to serving as a conventional coincidence detector, we demonstrate here that NMDA receptors can be activated under normal conditions, and such activation contributes to spiking properties of cingulate neurons. Using selective genetic knockouts of AC1, we demonstrated that calcium-calmodulin (CaM)-stimulated AC1 is critical for the tonic modulation of spike responses in cingulate neurons by NMDA receptors. These findings are consistent with our recent reports^[33] that under physiological conditions, NMDA receptors indeed contribute to normal synaptic transmission within the ACC at temperatures close to physiological conditions. Furthermore, our recent in vivo recordings from freely moving mice provide further evidence to support the concept that NMDA receptors in the ACC are actually very active under normal physiological conditions^[39]. At synaptic potential, one possible explanation of the functional NMDA receptors in adult cingulate neurons is that the distal dendrites are depolarized under physiological conditions. The depolarized membrane thus allows the removal of magnesium blockade of the NMDA receptors.

3.1 NMDA receptors and spike generation

There are reports that NMDA receptors contribute to synaptic transmission and modulation in the cortex, hippocampus, and spinal cord^[34,40-42]. For example, in thalamocortical slices of adult rats, both NMDA and non-NMDA receptors contribute to excitatory synaptic transmission^[41]. NMDA receptor-mediated sensory synaptic responses were also reported in the spinal dorsal horn neurons of adult mice^[34]. Functional roles of NMDA receptors in synaptic transmission are further supported by in vivo pharmacological and behavioral observations. Inhibition of NMDA receptors by pharmacological NMDA receptor antagonists AP-5 or MK801 produced sensory and motor effects in freely moving rats, including antinociceptive effects and rotarod deficits^[8,28]. In clinical studies, inhibition of NMDA receptors with ketamine, dextromethorphan, and 3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP) caused side effects including hallucinations, dysphoria, and disturbances of cognitive and motor function^[43]. These findings consistently suggest that under physiological conditions, glutamate NMDA receptors may play an active role by contributing to synaptic transmission^[39], spike generation as well as well-known synaptic plasticity^[1,27,38].

3.2 Calcium-activated AC1

Different isoforms of adenylyl cyclases have been reported within the central nervous system^[36]. In addition to their distribution in the hippocampus and their involvement in behavioral memory and NMDA receptor-dependent LTP^[32,44,45], specific adenylyl cyclase isoforms are also reported in the spinal cord dorsal horn, thalamus, and cortex, where they may contribute to other functions such as sensory transmission and modulation^[15,46,47]. Among them, AC1 is one of the two CaM-stimulated adenylyl cyclases in the central nervous system. The other is AC8. AC1 couples NMDA receptor-induced cytosolic Ca²⁺ increases to cAMP signaling pathways^[44] and the stimulation of Gs-coupled receptors^[48], while AC8 is mainly acting as a calcium sensor. In a previous report^[27], AC1 and AC8 contribute differently to injury-induced allodynia. AC1 deletion caused significant reduction in allodynia while AC8 deletion alone had no effect. This difference may result from AC1 and AC8 exhibiting different affinities for Ca^2+[36]. Considering AC1 is four to five times more sensitive to Ca²⁺ than AC8, it is reasonable to predict the involvement of AC1 in NMDA receptor-mediated effects. In the present study, we found that genetic deletion of AC1 blocked the NMDA receptor-dependent components of tonic spikes. It is likely that physiological levels of calcium in the ACC neurons are sufficient to generate basal levels of cAMP for contributing to the generation of action potentials.

3.3 ACC and pain

The ACC has been suggested to contribute to the perception of pain, to the learning processes associated with the prediction and avoidance of noxious sensory stimuli, as well as to pathological phantom pain^{[1,2,23,49,50]}. Previous studies from animals and humans demonstrate that the ACC neurons play key roles in behavioral nociceptive responses to injury in animals and pain perception or unpleasantness in humans. In humans, results from electrophysiological recordings from the ACC and functional imaging studies show that the ACC neurons respond to noxious stimuli^{[19,20,51-53]}. In animals, the ACC neurons respond to peripheral noxious stimuli or electrical shocks at high intensities^[9,27,54,55]. It is proposed that activity in the ACC may underlie the unpleasantness or discomfort associated with some somatosensory stimuli. Consistently, lesions in the ACC can reduce chronic pain in patients^[56]. In animal models of acute pain and persistent pain, lesions of the ACC produce antinociceptive effects^[4,57]. In freely moving animals, local administration of various opioid receptor agonists in the ACC produces powerful antinociceptive effects^[4]. Genetic deletion of AC1 and AC8 led to a complete abolishment of the behavioral allodynia caused by tissue injury and inflammation. These results support a role of the ACC in the processing of pain-related information. Furthermore, inhibition of NMDA receptor functions in the ACC also reduces pain-related fear memory^[45].

Although the ACC neuronal activity has been frequently reported in human imaging studies, the synaptic and cellular mechanisms for such higher order functions remain to be explored^[1]. Despite the seemly diffused physiological functions of ACC, there is strong physiological and pathological evidence indicating the importance of ACC activity in nociception, pain and persistent pain. Unlike studies of other cingulate-related functions that mostly remain at the level of investigating spike coding and activity, we were able to investigate at molecular and synaptic levels for possible mechanisms contributing to plastic changes in the ACC after the injury. For example, activation of NMDA receptors is critical for synaptic potentiation (or LTP) in cingulate neurons^[32]; subsequent activation of AC1 is important for such potentiation. Inhibition of NMDA receptors or genetic deletion of adenylyl cyclases including AC1 leads to a blockade of LTP and inhibition of behavioral sensitization (allodynia) to non-noxious stimuli after inflammation or nerve injury^[15,39]. Furthermore, injury including inflammation affecting the function of NMDA NR2B subtype receptors in adult cingulate neurons^[39], and inhibition of NR2B reduced behavioral allodynia. Our recent electrophysiological experiments using selective NR2B antagonist showed that NR2B contributed to NMDA receptor functions in adult cingulate neurons and inhibition of NR2B receptor-reduced LTP in the ACC (Wu et al., 2005). Our present studies provide evidence for a new role of NMDA receptors in cingulate neurons.

In summary, we present strong evidence for the contribution of NMDA receptors to the generation of ACC spikes. Therefore, there are at least three major roles of NMDA receptors in cingulate neurons: (1) a key coincident detector of synaptic transmission; (2) mediate normal synaptic responses; (3) contribute to spike responses. We propose that NMDA receptor-mediated synaptic responses may play important roles under these abnormal conditions and understanding the roles of NMDA receptors in synaptic transmission, regulation, and plasticity in the ACC may help us treat patients with different types of mental disorders.

REFERENCES

1 Zhuo M. Central inhibition and placebo analgesia. Mol Pain 2005; 1: 21. [PubMed abstract]

2 Zhuo M. Canadian Association of Neuroscience review: Cellular and synaptic insights into physiological and pathological pain. EJLB-CIHR Michael Smith Chair in Neurosciences and Mental Health lecture. Can J Neurol Sci 2005; 32(1): 27-36. [PubMed abstract]

3 Vogt BA. Pain and emotion interactions in subregions of the cingulate gyrus. Nat Rev Neurosci 2005; 6(7): 533-544. [PubMed abstract]

4 Lee DE, Kim SJ, Zhuo M. Comparison of behavioral responses to noxious cold and heat in mice. Brain Res 1999; 845(1): 117-121. [PubMed abstract]

5 Johansen JP, Fields HL, Manning BH. The affective component of pain in rodents: direct evidence for a contribution of the anterior cingulate cortex. Proc Natl Acad Sci USA 2001; 98(14): 8077-8082. [PubMed abstract]

6 Foltz EL, White LE Jr. Pain 搑elief?by frontal cingulumotomy. J Neurosurg 1962; 19: 89-100. [PubMed abstract]

7 Hurt RW, Ballantine HT Jr. Stereotactic anterior cingulate lesions for persistent pain: a report on 68 cases. Clin Neurosurg 1974; 21: 334-351. [PubMed abstract]

8 Zhuo M. Glutamate receptors and persistent pain: targeting forebrain NR2B subunits. Drug Discov Today 2002; 7(4): 259-267. [PubMed abstract]

9 Sikes RW, Vogt BA. Nociceptive neurons in area 24 of rabbit cingulate cortex. J Neurophysiol 1992; 68(5): 1720-1732. [PubMed abstract]

10 Vogt BA SR, Vogt LJ. Anterior cingulate cortex and the medical pain system. In 揘eurobiology of cingulate cortex and limbic thalamus: a comprehensive handbook? Vogt BA GM. ed. Boston: Birkhauser, 1993, 313-344.

11 Traub RJ, Silva E, Gebhart GF, Solodkin A. Noxious colorectal distention induced-c-Fos protein in limbic brain structures in the rat. Neurosci Lett 1996; 215(3): 165-168. [PubMed abstract]

12 Hutchison WD, Davis KD, Lozano AM, Tasker RR, Dostrovsky JO. Pain-related neurons in the human cingulate cortex. Nat Neurosci 1999; 2(5): 403-405. [PubMed abstract]

13 Wei F, Zhuo M. Potentiation of sensory responses in the anterior cingulate cortex following digit amputation in the anaesthetised rat. J Physiol 2001; 532(Pt 3): 823-833. [PubMed abstract]

14 Wei F, Li P, Zhuo M. Loss of synaptic depression in mammalian anterior cingulate cortex after amputation. J Neurosci 1999; 19(21): 9346-9354. [PubMed abstract]

15 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(4): 713-726. [PubMed abstract]

16 Kuo CC, Yen CT. Comparison of anterior cingulate and primary somatosensory neuronal responses to noxious laser-heat stimuli in conscious, behaving rats. J Neurophysiol 2005; 94(3): 1825-1836. [PubMed abstract]

17 Rainville P, Duncan GH, Price DD, Carrier B, Bushnell MC. Pain affect encoded in human anterior cingulate but not somatosensory cortex. Science 1997; 277(5328): 968-971. [PubMed abstract]

18 Rainville P. Brain mechanisms of pain affect and pain modulation. Curr Opin Neurobiol 2002; 12(2): 195-204. [PubMed abstract]

19 Talbot JD, Marrett S, Evans AC, Meyer E, Bushnell MC, Duncan GH. Multiple representations of pain in human cerebral cortex. Science 1991; 251(4999): 1355-1358. [PubMed abstract]

20 Vogt BA, Derbyshire S, Jones AK. Pain processing in four regions of human cingulate cortex localized with co-registered PET and MR imaging. Eur J Neurosci 1996; 8(7): 1461-1473. [PubMed abstract]

21 Casey KL. Forebrain mechanisms of nociception and pain: analysis through imaging. Proc Natl Acad Sci USA 1999; 96(14): 7668-7674. [PubMed abstract]

22 Calejesan AA, Kim SJ, Zhuo M. Descending facilitatory modulation of a behavioral nociceptive response by stimulation in the adult rat anterior cingulate cortex. Eur J Pain 2000; 4(1): 83-96. [PubMed abstract]

23 Tang J, Ko S, Ding HK, Qiu CS, Calejesan AA, Zhuo M. Pavlovian fear memory induced by activation in the anterior cingulate cortex. Mol Pain 2005; 1(1): 6. [PubMed abstract]

24 Bliss TV, Collingridge GL. A synaptic model of memory: long-term potentiation in the hippocampus. Nature 1993; 361(6407): 31-39. [PubMed abstract]

25 Collingridge GL, Bliss TV. Memories of NMDA receptors and LTP. Trends Neurosci 1995; 18(2): 54-56. [PubMed abstract]

26 Monyer H, Burnashev N, Laurie DJ, Sakmann B, Seeburg PH. Developmental and regional expression in the rat brain and functional properties of four NMDA receptors. Neuron 1994; 12(3): 529-540. [PubMed abstract]

27 Wei F, Wang GD, Kerchner GA, Kim SJ, Xu HM, Chen ZF, Zhuo M. Genetic enhancement of inflammatory pain by forebrain NR2B overexpression. Nat Neurosci 2001; 4(2): 164-169. [PubMed abstract]

28 Boyce S, Wyatt A, Webb JK, O扗onnell R, Mason G, Rigby M, Sirinathsinghji D, Hill RG, Rupniak NM. Selective NMDA NR2B antagonists induce antinociception without motor dysfunction: correlation with restricted localisation of NR2B subunit in dorsal horn. Neuropharmacology 1999; 38(5): 611-623. [PubMed abstract]

29 Tao YX, Rumbaugh G, Wang GD, Petralia RS, Zhao C, Kauer FW, Tao F, Zhuo M, Wenthold RJ, Raja SN, Huganir RL, Bredt DS, Johns RA. Impaired NMDA receptor-mediated postsynaptic function and blunted NMDA receptor-dependent persistent pain in mice lacking postsynaptic density-93 protein. J Neurosci 2003; 23(17): 6703-6712. [PubMed abstract]

30 Bliss TV. Young receptors make smart mice. Nature 1999; 401(6748): 25-27. [PubMed abstract]

31 Tang YP, Shimizu E, Dube GR, Rampon C, Kerchner GA, Zhuo M, Liu G, Tsien JZ. Genetic enhancement of learning and memory in mice. Nature 1999; 401(6748): 63-69. [PubMed abstract]

32 Liauw J, Wu LJ, Zhuo M. Calcium-stimulated adenylyl cyclases required for long-term potentiation in the anterior cingulate cortex. J Neurophysiol 2005; 94(1): 878-882. [PubMed abstract]

33 Liauw J, Wang GD, Zhuo M. NMDA receptors contribute to synaptic transmission in anterior cingulate cortex of adult mice. Acta Physiol Sin (生理学报) 2003; 55(4): 373-380. [PubMed abstract] [Full-text PDF]

34 Wang GD, Zhuo M. Synergistic enhancement of glutamate-mediated responses by serotonin and forskolin in adult mouse spinal dorsal horn neurons. J Neurophysiol 2002; 87(2): 732-739. [PubMed abstract]

35 Wang CC, Shyu BC. Differential projections from the mediodorsal and centrolateral thalamic nuclei to the frontal cortex in rats. Brain Res 2004; 995(2): 226-235. [PubMed abstract]

36 Xia Z, Storm DR. Calmodulin-regulated adenylyl cyclases and neuromodulation. Curr Opin Neurobiol 1997; 7(3): 391-396. [PubMed abstract]

37 Villacres EC, Wong ST, Chavkin C, Storm DR. Type I adenylyl cyclase mutant mice have impaired mossy fiber long-term potentiation. J Neurosci 1998; 18(9): 3186-3194. [PubMed abstract]

38 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(6): 573-579. [PubMed abstract]

39 Wu LJ, Toyoda H, Zhao MG, Lee YS, Tang J, Ko SW, Jia YH, Shum FW, Zerbinatti CV, Bu G, Wei F, Xu TL, Muglia LJ, Chen ZF, Auberson YP, Kaang BK, Zhuo M. Upregulation of forebrain NMDA NR2B receptors contributes to behavioral sensitization after inflammation. J Neurosci 2005; 25(48): 11107-11116. [PubMed abstract]

40 Sah P, Hestrin S, Nicoll RA. Tonic activation of NMDA receptors by ambient glutamate enhances excitability of neurons. Science 1989; 246(4931): 815-818. [PubMed abstract]

41 Gil Z, Amitai Y. Adult thalamocortical transmission involves both NMDA and non-NMDA receptors. J Neurophysiol 1996; 76(4): 2547-2554. [PubMed abstract]

42 Schiller J, Major G, Koester HJ, Schiller Y. NMDA spikes in basal dendrites of cortical pyramidal neurons. Nature 2000; 404(6775): 285-289. [PubMed abstract]

43 Kemp JA KJ, Gill R. NMDA receptor antagonists and their potential as neuroprotective agents. Handbook of Experimental Pharmacology 141: Ionotropic Glutamate Receptors in the CNS. Berlin: Springer-Verlag, 1999.

44 Wong ST, Athos J, Figueroa XA, Pineda VV, Schaefer ML, Chavkin CC, Muglia LJ, Storm DR. Calcium-stimulated adenylyl cyclase activity is critical for hippocampus-dependent long-term memory and late phase LTP. Neuron 1999; 23(4): 787-798. [PubMed abstract]

45 Zhao MG, Toyoda H, Lee YS, Wu LJ, Ko SW, Zhang XH, Jia Y, Shum F, Xu H, Li BM, Kaang BK, Zhuo M. Roles of NMDA NR2B subtype receptor in prefrontal long-term potentiation and contextual fear memory. Neuron 2005; 47(6): 859-872. [PubMed abstract]

46 Xia Z, Choi EJ, Wang F, Blazynski C, Storm DR. Type I calmodulin-sensitive adenylyl cyclase is neural specific. J Neurochem 1993; 60(1): 305-311. [PubMed abstract]

47 Xia ZG, Refsdal CD, Merchant KM, Dorsa DM, Storm DR. Distribution of mRNA for the calmodulin-sensitive adenylate cyclase in rat brain: expression in areas associated with learning and memory. Neuron 1991; 6(3): 431-443. [PubMed abstract]

48 Defer N, Best-Belpomme M, Hanoune J. Tissue specificity and physiological relevance of various isoforms of adenylyl cyclase. Am J Physiol Renal Physiol 2000; 279(3): F400-F416. [PubMed abstract]

49 Devinsky O, Morrell MJ, Vogt BA. Contributions of anterior cingulate cortex to behaviour. Brain 1995; 118 (Pt 1): 279-306. [PubMed abstract]

50 Johansen JP, Fields HL. Glutamatergic activation of anterior cingulate cortex produces an aversive teaching signal. Nat Neurosci 2004; 7(4): 398-403. [PubMed abstract]

51 Craig AD, Reiman EM, Evans A, Bushnell MC. Functional imaging of an illusion of pain. Nature 1996; 384(6606): 258-260. [PubMed abstract]

52 Davis KD, Taylor SJ, Crawley AP, Wood ML, Mikulis DJ. Functional MRI of pain- and attention-related activations in the human cingulate cortex. J Neurophysiol 1997; 77(6): 3370-3380. [PubMed abstract]

53 Derbyshire SW, Vogt BA, Jones AK. Pain and Stroop interference tasks activate separate processing modules in anterior cingulate cortex. Exp Brain Res 1998; 118(1): 52-60. [PubMed abstract]

54 Koyama T, Tanaka YZ, Mikami A. Nociceptive neurons in the macaque anterior cingulate activate during anticipation of pain. Neuroreport 1998; 9(11): 2663-2667. [PubMed abstract]

55 Li P, Kerchner GA, Sala C, Wei F, Huettner JE, Sheng M, Zhuo M. AMPA receptor-PDZ interactions in facilitation of spinal sensory synapses. Nat Neurosci 1999; 2(11): 972-977. [PubMed abstract]

56 Yarnitsky D, Barron SA, Bental E. Disappearance of phantom pain after focal brain infarction. Pain 1988; 32(3): 285-287. [PubMed abstract]

57 Pastoriza LN, Morrow TJ, Casey KL. Medial frontal cortex lesions selectively attenuate the hot plate response: possible nocifensive apraxia in the rat. Pain 1996; 64(1): 11-17. [PubMed abstract]