Acta Physiologica Sinica, August 25, 2003, 55(4): 373－380

Received 2003-04-08 Accepted 2003-05-12

Research supported by grants from NINDS (NS38680), NIDA (DA10833), the McDonnell Center for Higher Brain Function at Washington University, and the EJLB-CIHR Michael Smith Chair in Neurosciences and Mental Health.

Correspondence and requests for materials should be addressed to

Dr. Min Zhuo,

Department of Physiology, University of Toronto,

1 King's College Circle, Toronto,

Ontario M5S 1A8, Canada.

Tel: +1-416-978-4018;

Fax: +1-416-978-4940;

E-mail: min.zhuo@utoronto.ca

Research Paper

NMDA receptors contribute to synaptic transmission in anterior cingulate cortex of adult mice

Jason LIAUW1,Guo-Du WANG1, Min ZHUO1,2,*

1Departments of Anesthesiology, Anatomy & Neurobiology and Psychiatry, Washington

University Pain Center, Washington University, St. Louis, MO63110, USA;

2Department of Physiology, University of Toronto, Toronto, Ontario M5S 1A8, Canada

Abstract: Glutamatergic synapses are common excitatory chemical connections in mammalian central nervous system. At these synapses, most of baseline synaptic transmission is mediated by glutamate AMPA receptors. NMDA receptors that are sensitive to voltage-dependent magnesium blockade selectively contribute to activity-dependent synaptic plasticity. However, inhibition of NMDA receptors by systemic or local administration of NMDA receptor antagonists produced significant effects on different physiological functions that are not believed to depend on NMDA receptor related synaptic plasticity. Here we show that NMDA receptors contribute to synaptic responses in the anterior cingulate cortex (ACC), a region important for cognitive and other brain functions. The contribution of NMDA receptors became more prominent when synapses are stimulated at higher frequencies. Furthermore, at temperatures more close to physiological brain temperatures, more NMDA receptor mediated responses were recorded as compared to the room temperature. These data suggest a new function for NMDA receptors in the ACC as important postsynaptic receptors involved in synaptic transmission, in particular when cells are firing at high frequencies.

Key words: glutamate; anterior cingulate cortex (ACC); pain; mouse

NMDA受体参与小鼠的前额扣带回的神经突触传递

Jason LIAUW1,王过渡1, 卓敏1,2,*

1华盛顿大学医学院麻醉系, 解剖及神经生物系, 精神病学系, 华盛顿大学痛觉中心, 圣路易, 密苏里州 63110 美国;

2多伦多大学生理系, 多伦多大学痛觉研究中心, 多伦多, M5S 1A8 加拿大

摘要:谷氨酸性突触是哺乳动物神经系统的主要兴奋性突触。在正常条件下, 大多数的突触反应是由谷氨酸的AMPA受体传递的。NMDA受体在静息电位下为镁离子抑制。在被激活时, NMDA受体主要参与突触的可塑性变化。但是, 许多NMDA受体拮抗剂在全身或局部注射时能产生行为效应, 提示NMDA受体可能参与静息状态的生理功能。此文中, 我们在离体的前额扣带回脑片上进行电生理记录, 发现NMDA受体参与前额扣带回的突触传递。在重复刺激或近于生理性温度时, NMDA受体传递的反应更为明显。本文直接显示了NMDA受体参与前额扣带回的突触传递, 并提示NMDA受体在前额扣带回中起着调节神经元兴奋的重要作用。

关键词:谷氨酸; 前额扣带回; 痛觉; 小鼠

中图分类号:Q421

Among many neurotransmitters, glutamate is the major neurotransmitter mediating excitatory transmission in synapses throughout the central nervous system[1－3]. Glutamate mediates synaptic transmission by binding to postsynaptic AMPA (α-amino-3-hydroxy-5-methyl-isoxozole propionic acid), NMDA (N-methyl-D-aspartate), and KA (kainate) receptors[3,4]. While in most cases, synaptic responses are primarily mediated through postsynaptic AMPA and KA receptors, since NMDA receptors are blocked by magnesium at resting membrane potential. However, there are reports that NMDA receptors contribute to synaptic transmission and modulation in the cortex, hippocampus and spinal cord[5－8]. For example, in thalamocortical slices of adult rats, both NMDA and non-NMDA receptors contribute to excitatory synaptic transmission[6]. NMDA receptor mediated sensory synaptic responses were also reported in spinal dorsal horn neurons of adult mice[8]. Functional roles of NMDA receptors in synaptic transmission are further supported by in vivo pharmacological observations. Inhibition of NMDA receptors by pharmacological NMDA receptor antagonists AP5 or MK801 produced sensory and motor effects in freely moving rats, including antinociceptive effects and rotarod deficits[9,10]. Clinically, it is documented that 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[11]. Taken together, these studies suggest that in physiological conditions, glutamate NMDA receptors may play an active role, in addition to contributing behavioral learning and memory and neuronal death.

The anterior cingulate cortex (ACC) is a forebrain region thought to be important for cognitive functions of the brain, including attention, memory, emotion as well as sensory perception[10,12－18]. Genetic enhancement of NMDA receptor function in forebrain structures, including the hippocampus and ACC, improves learning and memory in mice, and also intensifies behavioral responses to tissue inflammation[19,20]. In the CA1 region of the hippocampus, enhanced NMDA receptor function leads to selective enhancement of synaptic potentiation or long-term potentiation (LTP) without significant contribution to basal synaptic transmission[21]. In the ACC, NMDA receptors are highly expressed, although their physiological function remains unclear[20]. Here we present evidence that in the ACC, NMDA receptors contribute to baseline postsynaptic responses in layer Ⅱ/Ⅲ evoked by single electrical shocks delivered to layer Ⅴ or to projecting fibers in the corpus callosum. These NMDA receptor mediated responses become more prominent at higher bath temperatures, suggesting the possible important roles of NMDA receptors in ACC related physiological functions.

1 METHODS

1.1 Slice preparation.

Adult male C57BL/6j (8－14 weeks old, Jackson) mice were anesthetized with 1－2% halothane. Transverse slices of ACC and hippocampus were prepared rapidly and maintained in an interface chamber at 28℃, where they were subfused with artificial cerebrospinal fluid (ACSF) consisting of (in mM) 124 NaCl, 4.4 KCl, 2.0 CaCl2, 1.0 MgSO4, 25 NaHCO3, 1.0 Na2HPO4, and 10 glucose, bubbled with 95% O2 and 5% CO2. Slices were kept in the recording chamber for at least two hours before experiments.

1.2 Electrophysiology.

In the ACC, a bipolar tungsten stimulating electrode was placed in layer Ⅴ, and extracellular field potentials were recorded using a glass microelectrode (3－12 MΩ, filled with ACSF) placed in layer Ⅱ/Ⅲ[22]. For hippocampal slices, a bipolar tungsten stimulating electrode was placed in the striatum radiatum in the CA1 region, and extracellular field potentials were recorded using a glass microelectrode (3－12 MΩ filled with ACSF) also in the striatum radiatum[20]. Stimulation at different intensities was applied, and synaptic responses were elicited at 0.02 Hz. To isolate synaptic responses mediated by different receptors, CNQX (20 μM) and AP5 (100 μM) were used to block AMPA/KA and NMDA receptor mediated responses, respectively. To test temperature-dependent responses, in some experiments, bath temperature was gradually decreased to 24℃ or increased to 36℃. Synaptic responses and bath temperature were continuously monitored and recorded.

In some experiments, intracellular recordings of synaptic responses were performed from neurons in the ACC with a 3 M potassium chloride-filled glass microelectrode (DC impedance 75－200 MΩ). The perfusion medium (in mM: NaCl 124; KCl 4; NaHCO3 26; NaH2PO4 1; MgSO4 1; CaCl2 2; glucose 10) 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. Bicuculline methiodide (10 μM) were added to the perfusion solution to block inhibitory transmission.

1.3 Data and statistical analysis.

Results were expressed as mean±standard error of the mean (S.E.M.). Statistical comparisons were performed with the use of one- or two-way analysis of variance (ANOVA) with the post-hoc Student-Newmann-Keuls test to identify significant differences.In all cases, P<0.05 was considered statistically significant.

Fig.1.

Slow field EPSPs recorded from adult mouse anterior cingulated cortex.

A: Diagram of a mouse ACC slice showing the placement of recording and stimulating (1, layer Ⅴ;

2, corpus callosum) electrodes.

B: Field EPSPs in the ACC recorded in normal ACSF in response to a brief stimulation (0.2 ms) delivered locally in the ACC. A fast EPSP induced by stimulation at a moderate intensity (left) and mixed fast and slow EPSPs induced by stimulation at greater intensity in the same slice (right).

C: Field EPSPs in the ACC recorded in normal ACSF in response to a brief stimulation (0.2 ms) delivered locally in the ACC (Layer V) or the corpus callosum. Note, the scale in C and D was greater than B in order to view the complete decay phase of slow EPSPs.

D: Field EPSPs recorded in the CA1 region of the hippocampus. Hippocampal slices were prepared from the same mice and incubated in the same chamber as ACC slices. Note that no slow response was observed in the hippocampus; each trace is the mean of three responses collected at 0.02 Hz.

E: NMDA receptor mediated EPSPs recorded intracellularly. Representative traces of EPSPs in control medium, 10 min after addition of CNQX (20 μM), 10 min after addition of AP5 (100 μM) and CNQX, and during washout with control medium.

2 RESULTS

Previous studies in ACC slices of rats showed that glutamate was the major fast excitatory transmitters and fast EPSCs or EPSPs were mediated by AMPA/KA receptors[22,23]. In ACC slices of adult mice, we found that a brief local electrical stimulation (0.2 ms duration) delivered to layer Ⅴ of the ACC (see Fig.1A), induced a fast EPSP. This finding is similar to results from adult rat ACC slices[22]. To determine if these fast field EPSPs are mediated by AMPA/KA receptors, we applied an AMPA/KA receptor antagonist CNQX (20 μM) through the bath solution. As in rats, this fast component was completely blocked by an AMPA/KA receptor antagonist CNQX (20 μM; n=10). In addition to fast field EPSPs, we observed a slow field EPSP when high intensity stimulation was used (n=20, see Fig. 1B). Slow EPSPs were intensity-dependent and stable when tested at 0.02 Hz. Slow field EPSPs per-sisted in the presence of CNQX (20 μM), suggesting that it is unlikely due to AMPA/KA receptor dependent polysynaptic responses.

Neurons in the ACC responded to stimulation of the callosal inputs in the corpus callosum[23,24]. Sah and Nicoll (1991) reported that both NMDA and non-NMDA receptors contribute to the callosal input induced EPSPs. To determine if similar results can be found in mouse ACC, we placed a bipolar stimulating electrode in the corpus callosum (see Electrode 2 in Fig.1A). Consistent with these results from rats, we found that electrical stimulation applied to the corpus callosum also induced fast and slow field EPSPs in the ACC slices (n=5; see Fig.1C for an example). We would like to point out that field EPSPs induced by local electrical stimulation may also activate the callosal inputs.

In the CA1 region of the hippocampus, it is well documented that field EPSPs are mainly mediated by AMPA receptors. To determine if NMDA receptor mediated responses may be due to technical differences in slice preparation, we decided to perform similar recordings from hippocampal slices obtained from the same mice kept under the same incubating conditions. In contrast to the results in the ACC, the same electrical shock delivered to Schaffer-collaterals induced only a fast synaptic response in the CA1 region of the hippocampal slices (Fig.1D); no slow responses were found in any slice tested (n=15). Consistent with the faster kinetics of EPSPs in the hippocampus, CNQX (20 μM) completely blocked synaptic potentials in that region, confirming that AMPA/KA receptors, but not NMDA receptors, underlie postsynaptic responses there (n=5). This finding indicates that postsynaptic receptors contributing to synaptic responses in the hippocampus may differ in composition from receptors in the ACC.

We next performed pharmacological experiments to examine if NMDA receptors contribute slow synaptic responses as previously revealed in rats[23]. Bath application of a NMDA receptor antagonist AP5 (100 μM) completely blocked the slow component of EPSPs (n=6; see Fig.2A for an example). The residual fast component was blocked by CNQX (20 μM) (Fig.2A). We also performed experiments by applying CNQX first followed by AP5. As expected, fast responses were selectively blocked by CNQX (n=10), while slow responses persisted and were later blocked by AP5 (n=6). Together these data confirm that slow EPSPs were mediated by NMDA receptors. To further investigate the slow synaptic responses, we performed intracellular recordings from ACC slices. We wanted to determine if slow EPSPs can be obtained from individual ACC neurons. A total of fifteen cells were recorded, and in most cases (n=12, 80%), we observed CNQX-insensitive EPSPs (see Fig.1E for an example). Application of a NMDA receptor antagonist AP5 (100 μM) blocked the residual EPSPs (Fig.1E), suggesting the involvement of NMDA receptors.

Fig.2.

Slow field EPSPs are mediated by glutamate NMDA receptors.

A: An example of field EPSPs recorded in control medium and 20 min after addition of AP5 (100

μM) and 30 min after addition of AP5 (100 μM) plus CNQX (20 μM);

B: The AP5-sensitive and CNQX-sensitive field EPSPs subtracted from traces in A;

C: Summation of NMDA mediated synaptic responses to a train of five pulses delivered at five different frequencies (5, 10, 20, 25 and 50 Hz).

One interesting feature of NMDA receptor-mediated responses is the temporal summation of their responses to repetitive stimulation. We tested if slow field EPSPs summate upon repetitive stimulation. Six pulses of stimuli were delivered at five different frequencies (5, 10, 20, 25 and 50 Hz). As shown in Fig.2C, synaptic responses summated at high frequencies between (10－50 Hz).

Finally, we wanted to determine if the temperatures of bath solution affect slow field EPSPs. After establish-ing baseline responses for at least 10 min, we stopped heating the bath chamber, and the bath temperature slowly decreased at 0.25℃/per min. At reduced bath temperatures, we found NMDA receptor mediated responses were significantly reduced, and the reduction was temperature-dependent (n=3; Fig.3). To evaluate changes in the slow responses at higher temperatures, we performed additional experiments and recorded responses at 30, 32 and 34℃. After establishing stable baseline responses at 28℃, we started to warm up the bath solution to higher temperatures. As shown in Fig.3A and B, slow field EPSPs were significantly increased at 30, 32 and 34℃ as compared to baseline responses at 28℃ (n=4). In some experiments (n=3), we also increased the temperature to 36－38℃. We found that electrical stimulation produced spike responses in addition to slow field EPSPs.

Fig.3.

NMDA receptor mediated synaptic responses are temperature-dependent.

A: Top, field EPSPs in the ACC recorded in normal ACSF at 28 and 24℃, each trace is the mean of three responses. Bottom, field EPSPs in the ACC recorded in normal ACSF at 28 and 34℃, each trace is the mean of three responses.

B: Summarized data for slow field EPSPs at different temperatures (n=3－4).

*P<0.05 vs field EPSPs at 28℃.

3 DISCUSSION

Our results provide a novel function for NMDA receptors as contributors to baseline excitatory transmission in the ACC. This role is selective for NMDA receptors in the ACC as opposed to the hippocampus, where only AMPA and KA receptors contribute to postsynaptic potentials. In addition, NMDA receptor-mediated EPSPs became more prominent at temperatures more close to the physiological temperature of the brain. The temperature-dependent enhancement of slow EPSPs in the ACC suggest that excitatory synaptic transmission are highly sensitive to temperature in this brain area. Unlike the ACC, however, synaptic responses in the hippocampus do not show similar temperature-dependent slow responses. In preliminary studies of in vivo recording from the adult rat and mouse ACC, we also observed a slow component field response to electrical stimulation delivered to the hindpaw (Wei, Tang and Zhuo, unpublished data). One possible important function for NMDA receptor mediated slow EPSPs in the ACC is that NMDA receptors would more likely contribute to synaptic transmission, integration and plasticity at abnormal higher brain temperatures under pathological conditions. Furthermore, due to their summation properties, these NMDA receptor-mediated synaptic responses are likely to play a significant role in ACC neuronal excitability, possibly affecting patterns of action potential firing. Our results thus provide strong evidence that NMDA receptors play important roles in the physiological functions of the ACC neurons under normal or pathological conditions.

Recent studies from humans and animals consistently suggest that the ACC play an important role in emotional and attentive responses to internal and external stimulation[16,25,26]. Neuroimaging and electrophysiological studies in humans have shown that somatosensory stimuli, including those causing pain, activate several limbic areas, including the ACC[13,27－32]. Electrophysiological recording experiments demonstrate that ACC neurons respond to peripheral nociceptive stimulation in animals[32－35]. Recent behavioral experiments in both rats and mice show that lesion of the medial frontal cortex, which includes the ACC, significantly reduced animals' sensitivity to noxious heat applied to the hindpaw in the hot-plate test[36,37]. Electrical and chemical stimulation of regions within the ACC facilitated behavioral responses to noxious heat in the tail-flick test[38]. Consistent with these animal studies, the unpleasantness of pain is abolished in patients with frontal lobotomies or cingulotomies[39－41]. As compared with sensory neurons in the spinal cord, most ACC neurons show receptive fields receiving input from almost any part of the body surface[13,34] (Zhuo et al., unpublished data). Furthermore, most ACC neurons are polymodal (e.g., responding to both non-noxious and noxious stimuli). In monkeys, it has also been reported that some nociceptive ACC neurons were activated during anticipation of pain (before actual noxious stimuli)[33]. It has been proposed that neurons in ACC may contain little or no coding for the location of noxious stimuli on the body surface but contribute the affective content of noxious stimuli or unpleasantness of pain[25,42,43].

Just like other regions in the central nervous system, fast excitatory synaptic transmission within the ACC is mediated by the excitatory amino acid glutamate[22,23,44]. Evoked EPSPs or EPSCs recorded using intracellular or whole-cell patch-clamp techniques were mediated through postsynaptic AMPA/KA receptors[22,23,44]. Consistent with these previous reports, our present results showed that fast synaptic responses are mediated by glutamate AMPA/KA receptors in the ACC of adult mouse. Furthermore, NMDA receptors can also contribute to synaptic responses, especially at temperatures closer to physiological temperatures.

NMDA receptors are highly expressed in the ACC, including NR1, NR2A and NR2B subtype receptors[20,45－47]. Activation of NMDA receptors is known to contribute to synaptic plasticity such as LTP[21]. In the ACC, theta burst stimulation induced long-lasting potentiation of synaptic responses, while repetitive, prolonged low-frequency stimulation (1 Hz for 15 min) produced depression[22,49].

Pharmacological experiments showed that both L-type voltage-gated calcium channels (L-VDCCs) and metabotropic glutamate receptors (mGluRs) are required for inducing LTD, whereas NMDA receptor activation is not[22]. Our recent preliminary studies found that inhibition of NMDA receptors significantly reduced LTP induced by theta burst stimulation in the ACC (unpublished data). Thus, it is most likely that NMDA receptors selectively contribute to synaptic potentiation in the ACC.

The present study may also broaden the known range of physiological functions for NMDA receptors in the mammalian forebrain areas. In addition to serving as a coincidence detector, NMDA receptors contribute significantly to synaptic transmission in the ACC. Some of behavioral deficits in previous studies using NMDA subtype receptor overexpression or deletion in mice may be due to changes in NMDA receptor mediated synaptic responses in the ACC, since neurons in the ACC have been consistently implicated in different functions of the brain such as attention, memory and persistent pain[10,16,20,48]. Because NMDA receptors are coupled to various second messenger signaling pathways, activation of NMDA receptors during normal synaptic transmission will provide a background level of activity of these intracellular signaling pathways.

Finally, our results may help explain some of the unexplained side-effects of NMDA receptor antagonists in humans, which include psychotomimetic effects such as sedation, psychosis and hallucinations[11]. Furthermore, in pathological conditions such as persistent inflammation, the up-regulation of NMDA receptors was observed in some areas of the forebrains (Qiu et al., in preparation). We suggest that NMDA receptor-mediated synaptic responses may play important roles in these abnormal conditions and understanding the roles of NMDA receptors in synaptic transmission, regulation and plasticity in the ACC may help us to treat patients with different types of neuronal disorders.

***

Acknowledgement: We would like to thank members of Zhuo lab for helpful discussions.

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