A Synaptic Model for Pain: Long-Term Potentiation in the Anterior Cingulate Cortex

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1 Mol. Cells, Vol. 23, No. 3, pp Minireview Molecules and Cells KSMCB 2007 A Synaptic Model for Pain: Long-Term Potentiation in the Anterior Cingulate Cortex Min Zhuo* Department of Physiology, Faculty of Medicine, University of Toronto Center for the Study of Pain, University of Toronto, Toronto, Ontario M5S 1A8, Canada. (Received February 25, 2007; Accepted February 27, 2007) Investigation of molecular and cellular mechanisms of synaptic plasticity is the major focus of many neuroscientists. There are two major reasons for searching new genes and molecules contributing to central plasticity: first, it provides basic neural mechanism for learning and memory, a key function of the brain; second, it provides new targets for treating brain-related disease. Long-term potentiation (LTP), mostly intensely studies in the hippocampus and amygdala, is proposed to be a cellular model for learning and memory. Although it remains difficult to understand the roles of LTP in hippocampus-related memory, a role of LTP in fear, a simplified form of memory, has been established. Here, I will review recent cellular studies of LTP in the anterior cingulate cortex (ACC) and then compare studies in vivo and in vitro LTP by genetic/ pharmacological approaches. I propose that ACC LTP may serve as a cellular model for studying central sensitization that related to chronic pain, as well as painrelated cognitive emotional disorders. Understanding signaling pathways related to ACC LTP may help us to identify novel drug target for various mental disorders. Keywords: Adenylyl Cyclases; Anterior Cingulate Cortex; Fear Memory; Gene Knockout; Immediate Early Genes; Long-Term Depression; Long-Term Potentiation; Mice; Persistent Pain; Synaptic Plasticity. Introduction Studying synaptic changes in central synapses, such as long-term potentiation (LTP) of synaptic responses, is believed to be the key cellular basis for information process in the mammalian brains. Synaptic modifications are * To whom correspondence should be addressed. Tel: ; Fax: min.zhuo@utoronto.ca not only happening during physiological conditions, such as learning and memory, but also in pathological conditional conditions, including persistent pain, drug addiction and emotional disorders (Bliss and Collingridge, 1993; Ji et al., 2003; Kandel, 2001; Merzenich, 1998; Nicoll and Malenka, 1995; Zhuo, 2005). Furthermore, LTP is found in many central synapses where normal learning is unknown to occur. Thus, it is likely that LTP may serve as one of basic neuronal synapse function in mammalian brains. LTP has been reported in many brain areas including the cortex, hippocampus and spinal cord. Table 1 shows previous reports of LTP in several major sensoryrelated processing areas, such as sensory perception (prefrontal cortex (PFC), anterior cingulate cortex (ACC), transmission (spinal cord), modulation (ACC, periaqueductal gray (PAG), rostroventral medulla (RVM)) and memory (hippocampus, amygdala). Among them, the hippocampus is the mostly investigated brain area. Two major reason behind the investigation of the hippocampus, one is the clearly defined anatomic input pathways, and allow easy field recordings in the brain slices; second, its proposed role in behavioral memory tests. Indeed, many brain areas share similar signaling pathways with the hippocampus. However, it must be noted that there are also exceptions. Thus, it is wrong to assume that all brain synapses will work in the same way as hippocampal synapses. Studies from various central regions of the brain suggest that the induction protocol for LTP is not universally applicable. For example, strong tetanic stimulation that induces LTP in the hippocampus failed to induce LTP in many other areas of the brain; second, the signaling pathways contributing to LTP induction is not the same. Different subtypes of glutamate receptors as well as other transmitters reported to contribute to the induction of LTP; third, the physiological and/or pathological role of Abbreviations: ACC, anterior cingulate cortex; LTP, long-term potentiation.

2 260 A Synaptic Model for Pain: Long-Term Potentiation in the Anterior Cingulate Cortex Table 1. Long-term potentiation (LTP) recorded from sensoryrelated central synapses. Prefrontal frontal cortex (PFC) Anterior cingulate cortex (ACC) Induction protocol TBS Paired training Strong tetanic stimulation TBS Paired training Spike-timing protocol Insular cortex TBS LTP Somatosensory cortex TBS Hippocampus Strong tetanic stimulation TBS Paired training Spike-timing protocol amygdala Strong tetanic stimulation TBS Paired training Spike-timing protocol / Thalamus ND ND Periaqueductal gray (PAG) ND ND Rostroventral medulla (RVM) ND ND Spinal dorsal horn Strong tetanic stimulation TBS Paired training Spike-timing protocol / ND, significant potentiation;, no potentiation; /, potentiation only in some cases; ND, no data available LTP in different parts of the brain is still unclear. Among them, how LTP may contribute to cognitive learning and memory remains obscure, despite long-time efforts of numerous neuroscientists. In this article, I will review the mechanisms for LTP in the ACC, a key limbic structure for cognition and other brain functions, and explore possible functional significance of cingulate LTP in pain and pain-related cognitive functions. Anatomy of the ACC Anterior cigulate cortex (ACC) is the frontal part of the cingulate cortex in mammalian brains. Figure 1 shows illustrative structure of the ACC in a mouse brain section, and a modified drawing of projecting fibers and local neurons by Cajal (Fig. 1B). The ACC contains layers of pyramidal cells and mixed with local interneurons. In layer I, many projecting fibers are from other central nuclei, and branching into deeper layers in the ACC. The layers II-III contain mainly pyramidal cells. Neurons in layer II-III receive sensory inputs from the medial thalamus, a key relay nucleus for somatosensory information including A B Fig. 1. Diagram of the mouse anterior cingulate cortex (ACC). A. An unilateral coronal section the mouse brain containing the ACC and adjacent brain areas. cg1, cingulate cortex, area 1; cg2, cingulate cortex, area 2; gcc, genu corpus callosum; cg, cingulum; M2, secondary motor cortex; M1, primary motor cortex; S1, primary somatosensory cortex; Gi, granular insular cortex; LV, lateral ventricule; Cpu, caudate putamen (striatum). B. A modified copy of Cajal s drawing of 8-day-old mouse cingular cortex using the Golgi method. pain (Shibata, 1993; Wang and Shyu, 2004; Yamamura et al., 1996). Neurons in layer V are larger than cells in layers II-III and VI, also receive sensory inputs including noxious information. ACC neurons form interconnection with the other ACC neurons in the opposite side of the hemisphere through callosal projecting fibers, as well as other cortical areas at the same side and opposite side of the brains. Most ACC neurons respond to both noxious and nonnoxious stimuli. Many non-pyramidal cells are inhibitory neurons that contain GABA and/or neuropeptides. It typically shows reduced spike responses to peripheral noxious stimuli, while pyramidal cells show excitatory or increased spike responses. It is important to note that while the soma of the pyramidal cells may be strictly located at the layer V, its distal branches can extend to other layers of the ACC such as layer I. Thus, the evoked responses recorded from the layer V cells maybe contain synaptic

3 Min Zhuo 261 Table 2. A list of physiological and pathological functions/roles of the anterior cingulate cortex based on data from experimental animals, normal human and patient brain imaging studies. Sensory, Pain Functions Somatosensory pain Visceral pain Inflammatory pain Neuropathic pain Phantom pain Social pain/exclusion Imagined pain Chronic migraine Fig. 2. The interaction and balance between excitation and inhibition within the ACC. A diagram shows the excitatory (glutamate, Glu) and inhibitory (GABA) synapses to link pyramidal cells and inhibitory neurons within different layers of the ACC. responses occurring at outer layers of the ACC. Within the local circuits, inhibitory neurons often receive innervations from the pyramidal cells (glutamatergic), then release GABA onto the perisomatic region of the pyramidal cells (Fig. 2). Considering the difference in their morphology and the pattern of spike activities, pyramidal and inhibitory neurons can be identified electrophysiologically in brain slice preparations (Zhao et al., 2005b). Pyramidal cells in the layer V project to subcortical structures such as the hypothalamus and PAG, may contribute to descending modulation of spinal sensory transmission (for example, see Calejesan et al., 2000). ACC neurons also form interconnections with neurons in the amygdala, a structure critical for emotional fear and anxiety. This anatomic connection suggests possible critical roles of ACC in emotional fear, anxiety and depression. Furthermore, ACC neurons project to motor cortex to generate motor responses, vocalization etc. In particular, in humans and monkeys, spindle neurons have been reported in the layer V, and the loss of these spindle cells are affected in AD patients (Nimchinsky et al., 1995). Physiological and pathological importance of the ACC Unlike other nuclei, ACC neurons are multi-functional, at least superficially (Botvinick et al., 2004; Dalley et al., 2004; Peoples, 2002; Sanders et al., 2002). Studies from animals and humans consistently demonstrate that ACC neurons play important roles in various sensory, memory, Learning and memory Cognition Emotion Contextual fear memory Auditory fear memory Trace fear memory Avoidance memory Working memory Remote spatial memory Anticipation Attention Conflict monitoring Novelty detection Reward assessment, loss and gain Decision-making Anxiety Depression Romatic love Sex Panic disorder Bipolar disorder Schizophrenia emotion and cognitive functions. Using the PubMed searching, one can easily find the direct or indirect evidence for the involvement of ACC in a wide range of certain brain functions. The rapid increased knowledge of the ACC in higher brain functions is mainly due to the development of modern human brain imaging technique. Table 2 summarizes four major functions of the ACC: sensory perception; learning and memory, emotion, and cognitive functions. In this article, I mainly focus on the roles of ACC in sensory functions including pain. It is important to point out that human functional studies are often simply the correlation studies of brain activity with different testing conditions. No detailed investigation of cellular mechanisms can be performed in human imaging studies. Many conclusions can be easily switched to the opposite way, because most of these imaging studies failed to identify the direct source of neural activity (such as excitatory pyramidal vs local inhibitory neurons) (Zhuo, 2005). For sensory functions including pain, lesions of the ACC significantly reduced acute nociceptive responses,

4 262 A Synaptic Model for Pain: Long-Term Potentiation in the Anterior Cingulate Cortex and formalin injection induced aversive memory behaviors (Johansen et al., 2001; Lee et al., 1999). In patients with frontal lobotomies or cingulotomies, the unpleasantness of pain is abolished (see Zhuo, 2002 for review). Electrophysiological recordings from the ACC neurons found that neurons within the ACC respond to noxious stimuli, including nociceptive specific neurons (Hutchison et al., 1999; Sikes and Vogt, 1992). Neuroimaging studies further confirm these observations and show that the ACC, together with other cortical structures, are activated by acute noxious stimuli (Casey, 1999; Rainville et al., 1997; 2001; Talbot et al., 1991). Recent studies from healthy humans or patients suggest that ACC activity is also related to the empathy of pain, social exclusion/pain, chronic migraine as well as hypothesized pain (de Tommaso et al., 2005; Derbyshire et al., 2004; Eisenberger et al., 2003; Johansen and Fields, 2004; Koyama et al., 2001; Singer et al., 2004). Stimulation of ACC neurons by delivering electrical currents or local glutamate microinjection cause fear memory or trigger aversive behaviors, indicating that stimulation of the ACC is painful, fearful or aversive (Johansen and Fields, 2004; Lei et al., 2004; Tang et al., 2005). One special human case is the report of a patient with chronic headache in Korea. For unknown reason, this construction worker complained headache for years. After brain imaging examination, it was found that there was a nail deep in his brain, region related to the ACC and related cortical areas (Fig. 3). This case strongly supports the important role of the ACC and its related prefrontal cortex in pain. It has been noted that brain tumor in these areas often trigger spontaneous pain and headache. Fig. 3. A nail in the brain triggers chronic pain in a Korea construction worker. A human case report (by Yahoo news) of central pain triggered by a metal nail embedded in the forebrain. In Seoul, a construction worker complained pain for several years until the X-rays examination revealed that he had a two-inch nail embedded in his skull, within the area related to ACC and other forebrain areas. This is a very interesting human case to support the critical roles of ACC and related frontal areas in pain. tent pain related loss of trace fear memory (Zhao et al., 2006). Multiple functions of ACC neurons Most popular approaches of modern neuroscience is to identify a selective area for testing a selective behavioral functions. For example, the spinal dorsal horn is often used for studying sensory and pain, while the hippocampus is for spatial memory. However, the ACC seems to play important roles in a wide variety of behavioral functions, including sensory, memory, emotional and cognitive functions (see above). One simple approachable function for the ACC is pain and unpleasantness. The involvement of ACC in various functions of brains provide most powerful evidence that ACC indeed a key region of pain processing, and a region where pain interferes other higher brain functions, such as cognition, emotion and even consciousness. Progress has been made in related the ACC to pain (including persistent or chronic pain) and cognitive memory in animal studies (Chai et al., 2006; Frankland et al., 2004; Teixeira et al., 2006; see Zhuo, 2006 for review). For example, a recent study in mice reported that ACC plasticity may play a key role in persis- Integrative experimental approaches for ACC investigations Different preparations have been employed to investigate the molecular, cellular, physiological functions of the ACC, from in vitro cultured neurons, brain slice electrophysiology, to in vivo ACC recordings in freely moving animals, and behavioral tests. Combinations with transgenic and gene knockout mice provide powerful tool to connect molecular mechanism to behavioral functions in whole animals. Thus, understanding of synaptic mechanism within the ACC will greatly help us to gain insights into long-term plastic changes in the brain related to central pain. Synaptic transmission in the ACC Brain slices from mouse, rats, guinea pigs, and rabbit have been used to investigate synaptic transmission in the ACC (Higashi et al., 1991; Sah and Nicoll, 1991; Tanaka and North, 1994; Vogt and Gorman, 1982; Wei et al.,

5 Min Zhuo 263 Fig. 4. Excitatory and inhibitory synaptic transmission in the ACC. Presynaptic activity triggers vesicle releases in the ACC excitatory synapses. Fast excitatory transmission is mediated by glutamate in the ACC. Most of excitatory synaptic currents (EPSCs) are mediated by AMPA receptors, while small percentages of EPSCs are mediated by GluR6-containing KA receptors. In physiological temperature or in freely moving animals, synaptic responses are also mediated by postsynaptic NMDA receptors. Therefore, under in vivo conditions, different classes of glutamate receptors, including AMPA, KA and NMDA receptors, likely mediate excitatory synaptic transmission in the ACC. Inhibitory transmission is likely mediated by postsynaptic GABA A receptors. Unlike excitatory transmission, little work has been reported on the inhibitory systems in the ACC. A recent report showed that glutamate GluR5 containing KA receptor located at presynaptic terminals of inhibitory neurons and regulate the release of GABA. 1999; Wu et al., 2005; Zhao et al., 2005). Previous studies were mostly performed by intracellular or field recordings. Recent studies using whole-cell patch-clamp recordings from slices of adult mice provide key information for postsynaptic receptors contribute to synaptic transmission in mouse and rat ACC. Excitatory transmission AMPA receptor AMPA receptors, including GluR1 and GluR2/3 subunits, are found in the ACC neurons (Wei et al., 1999). Glutamate is the major fast excitatory transmitter in the ACC (Wei et al., 1999). This is nicely demonstrated by the observation that bath application of CNQX and D-2- amino-5-phosphonopentanoic-acid (AP-5) completely abolishes fast EPSCs recorded in the ACC neurons. Different types of glutamate receptors, including AMPA, kainate (KA), NMDA and metabotropic receptors (mglurs) are found in the ACC. Fast synaptic responses induced by focal stimulation are completely blocked by CNQX, a non-selective inhibitor of AMPA and KA receptors (Wei et al., 1999; Wu et al., 2005a) (Fig. 5). Kainate receptor KA receptors have been reported to Fig. 5. Signaling pathways for the induction and expression of synaptic LTP in the ACC. Neural activity triggered the release of excitatory neurotransmitter glutamate (Glu: filled circles) in the ACC synapses. Activation of glutamate NMDA receptors leads to an increase in postsynaptic Ca 2 in dendritic spines. Both NMDA NR2B and NR2A subunits are important for NMDA receptor functions. Ca 2 serves as an important intracellular signal for triggering a series of biochemical events that contribute to the expression of LTP. Ca 2 binds to CaM and leads to activation of calcium-stimulated ACs, mainly AC1 and Ca 2 /CaM dependent protein kinases (PKC, CaMKII and CaMKIV). Through various protein-kinase related intracellular signaling pathways, the trafficking of postsynaptic AMPA receptor as well as other synaptic modifications contribute to enhanced synaptic responses. Activation of CaMKIV, a kinase predominantly expressed in the nuclei, will trigger CREB signaling pathways. In addition, activation of AC1 and AC8 lead to activation of PKA, and subsequently CREB as well. MAPK/ERK could translocate from the cytosol to the nucleus and then regulate CREB activity. Considering the upstream promoter region of the Egr1 gene contains CRE sites, it is possible that Egr1 may contribute to CREB-related signaling targets in the ACC neurons. Considering the fact that many late signaling molecules (e.g., FMRP, Egr1 and CaMKIV) also contribute to early enhancement of responses, more works are needed to reveal the signaling pathways for the LTP in the ACC neurons. contribute to sensory transmission in the spinal cord (Li et al., 1999). Recent studies using whole-cell patch-clamp recordings from genetically modified mice show that postsynaptic KA receptors contribute to fast synaptic transmission in pyramidal neurons in the ACC. Single-shock stimulation could induce small KA receptor-me-diated excitatory postsynaptic currents (KA EPSCs) in the presence of picrotoxin, AP-5, and a selective AMPA receptor antagonist, GYKI KA EPSCs had a significantly slower rise time course and decay time constant compared with AMPA receptor-mediated EPSCs. High-frequency repetitive stimulation significantly facilitated the KA EPSCs (Wu et al., 2005a). Genetic deletion of the GluR6 or GluR5 subunit significantly reduced, and GluR5 and 6 double knockout completely abolished, KA EPSCs and KA-activated currents in ACC pyramidal neurons (Wu et al., 2005a). NMDA receptor Most of AMPA and KA receptors mediated responses are studied under experimental conditions

6 264 A Synaptic Model for Pain: Long-Term Potentiation in the Anterior Cingulate Cortex in in vitro brain slices. In adult ACC slices at physiological temperatures, NMDA receptor mediated slow synaptic responses were also recorded from the ACC in vitro brain slices and in vivo freely moving mice (Liauw et al., 2003; Wu et al., 2005b), suggesting that NMDA receptors are tonically active in this region. This finding is consistent with numerous reports of side-effects of NMDA receptor antagonist in whole animals under normal physiological conditions. Therefore, it is always important to remember that brain slice recordings sometimes do not mimic physiological conditions in vivo, including glutamate receptor mediated transmission. In addition, a recent report found that NMDA receptors in the ACC neurons contribute to spike responses in ACC neurons (Wang and Zhuo, 2006). Inhibitory transmission Besides excitatory transmission in the ACC, GABA is the major inhibitory transmitter in the ACC. Bath application of picrotoxin completely abolishes sipscs and eipscs. The IPSCs are mainly mediated postsynaptic GABA A receptors (Wu et al., 2006). GABA B receptors are also reported in the ACC, although the roles of GABA B have not been investigated. Unlike excitatory synaptic transmission, few studies have been reported about the inhibitory transmission. Recent studies using KA knockout mice reported that inhibitory transmission in the ACC under tonic modulation of KA GluR5 receptor modulation (Wu et al., 2006). Stimulation protocols for inducing LTP in the ACC Synaptic and cellular mechanisms of LTP in the ACC are best studied using brain slice preparations. Different stimulation protocols have been found for inducing longlasting potentiation of synaptic responses in the ACC cells. LTP of excitatory synapses responses can be recorded in the ACC using field recording and whole-cell patch-clamp recording techniques (Wei et al., 1999; Zhao et al., 2005b). For field recording from adult rat or mouse ACC slices, glutamatergic synapses in the ACC can undergo longlasting potentiation in response to theta burst stimulation (TBS), a paradigm more closely mimicking the activity of ACC neurons. The potentiation lasted for at least 40 to 120 min (Wei et al., 2002a). Unlike the hippocampus, strong tetanic stimulation in the ACC did not cause reliable longterm potentiation (Wei et al., unpublished data). Whole-cell patch-clamp recordings allow better investigation of synaptic mechanisms for LTP in the ACC (Zhao et al., 2005b). LTP can be induced using three different protocols, including the pairing training protocol, the spike-epsp pairing protocol, and TBS protocol (Zhao et al., 2005b). Unlike the field recordings, LTP induced by the pairing protocol is mainly triggered by the activation of NMDA receptors but not through L-type VDCCs (Zhao et al., 2005b). These studies provide clear evidence that LTP induced by different stimulation protocols may share some common signaling pathways. It is important to keep in mind that LTP recorded through field recording or whole-cell patch-clamp recording may employ different synaptic signaling pathways for induction and expression. The induction of ACC LTP Receptors/ion channels for the induction of cingulate LTP NMDA receptor NMDA receptors are well-known for the induction of LTP in many central synapses. In the ACC, the induction of LTP by different protocols is mostly NMDA receptor dependent. In the ACC, NMDA receptor containing NR2A or NR2B subunits contribute to most of NMDA receptor currents (Zhao et al., 2005b). Bath application of a NR2A antagonist NVP-AAM077 and NR2B antagonist ifenprodil/ro compounds produce almost completely blockade of NMDA receptor mediated EPSCs. Several recent studies reported the non-selective effects of NVP-AAM077 on NMDA NR2A vs NR2B receptors. However, most of these recordings are performed from in vitro cell lines that expressing cloned receptors. It is hard to known if such artificial made receptors may mimic NMDA receptors in adult ACC neurons. Furthermore, we found that NVP- AAM077 at a higher dose failed to abolish all RO- or ifenprodil sensitive currents (unpublished data). In the ACC, application of NR2A or NR2B antagonist reduces LTP, without complete abolishment of LTP. LTP only is abolished after the co-application of both inhibitors (Zhao et al., 2005b). It is noted that LTP induced by spike-timing protocol seems to more sensitive to NMDA NR2B blockade as compared with effects on LTP induced by pairing training protocol (Zhao et al., 2005b). L-type voltage-gated calcium channels (L-VDCCs) In addition to NMDA receptors, L-VDCCs are also required for inducing LTP (Liauw et al., 2005) when LTP is induced by TBS in field recording conditions. Together with the effects of NR2A and NR2B antagonists, it strongly suggests that LTP induced by different protocols in the ACC may mimic different physiological/pathological conditions by distinct signaling pathways. Future experiments are clearly needed to explore this possibility. Intracellular signaling pathways for required for synaptic potentiation Ca-CaM Activation of glutamate NMDA receptors leads to

7 Min Zhuo 265 Table 3. Stimulation protocols for inducing LTP in the ACC slices. Theta-burst stimulation Pairing training Spike-EPSP pairing (spike-timing) protocol Parameters Five trains of bursts (four pulses at 100 Hz) of stimuli delivered every 200 ms Five trains of bursts (four pulses at 100 Hz of stimuli delivered every 200 ms, repeated four times at an interval of 10 s Paired presynaptic 80 pulses at 2 Hz with postsynaptic depolarization at 30 mv Paired three presynaptic stimuli that caused three EPSPs (10 ms ahead) with three postsynaptic APs at 30 Hz, paired 15 times every 5 s Recording method Field potential recording Whole-cell patch-clamp recording Whole-cell patch-clamp recording Whole-cell patch-clamp recording an increase in postsynaptic Ca 2 in dendritic spines. Ca 2 serves as an important intracellular signal for triggering a series of biochemical events that contribute to the expression of LTP. Ca 2 binds to CaM and leads to activation of calcium-stimulated signaling pathways (Wei et al., 2003). Furthermore, postsynaptic injection of BAPTA completely blocked the induction of LTP, indicating the importance of elevated postsynaptic Ca 2 concentrations (Zhao et al., 2005b). A recent work using electroporation of mutant CaM in the ACC suggest that Calcium binding sites of CaM is critical for the induction of cingulate LTP (Wei et al., 2003). Adenylyls cyclases: camp related signaling pathways are the key signaling pathways in biological systems. Among more than 10 subunits, AC1 and AC8 are two AC subtypes that respond positively to calcium-cam (Xia and Storm, 1997). As compared with AC8, AC1 is more sensitive to calcium increase. In the ACC, AC1 is highly expressed in cingulate neurons located in most of layers (Wei et al., 2002b). AC1 is selective for plastic changes; gene deletion of AC1 does not affect basal glutamate transmission in the ACC. By contrast, LTP induced by TBS or pairing stimulation are abolished in cingulate pyramidal cells (Liauw et al., 2005). AC1 is also contributing to synaptic potentiation induced by forskolin, a AC activator that is non-selective for AC isoform. Gene deletion of AC8 subunit partially contributes to forskolininduced potentiation. Whole-cell patch-clamp recording also revealed that AC1 activity is required for the induction of LTP in ACC pyramidal cells. By using chemical design and biochemical screening, several selective inhibitors of AC1 has been identified. Consistently, pharmacological inhibition of AC1 in ACC neurons abolished LTP induced by pairing training (Zhuo, unpublished data). CaMKIV CaMKIV is a major neuronal signaling pathway by which Ca 2 activates CREB involves the Ca 2 /calmodulin dependent protein kinase type IV (CaM- KIV). CaMKIV is a member of a group of multifunctional CaMK. CaMKIV is distinguished among the CaM kinases in its capacity to activate CREB-dependent transcription both by virtue of its nuclear localization and catalysis of CREB phosphorylation on Serine 133. CaMKIV also promotes CREB function by activating the transcriptional co-activator CREB binding protein (CBP). CaMKIV is enriched in the ACC (Wei et al., 2002a). CaMKIV is required for CaM translocation triggered by neural activity into the nuclei of ACC neurons. Previous studies have shown that CaM translocation reflects the trapping of Ca 2 -CaM complexes by nuclear CaM binding proteins. The abolishment of CaM translocation in CaMKIV knockout mice identifies CaMKIV as the critical sink that traps Ca 2 -CaM complexes in neuronal nuclei. This trapping leads to CaMKIV activation and subsequent CREB phosphorylation and activation. Consistently, we found in both in vitro and in vivo conditions that activation of CREB were significantly reduced or abolished in CaM- KIV knockout mice. Considering the important roles of CREB in LTP, we found that synaptic potentiation induced by TBS were reduced or abolished in the same areas (Wei et al., 2002a). Gene expression and synaptic potentiation While the involvement of different protein kinases and second messengers in the ACC LTP are predicted, recent data of the requirement for several immediate early genes and gene-related proteins in ACC LTP are surprising. Potentiation induced in ACC synapses within min is affected by the gene deletion of Egr1 and FMRP. These effects are unlikely due to indirect inhibition of NMDA receptors, since NMDA receptor mediated currents are not affected. FMRP FMRP is a ubiquitously expressed mrna binding protein associated with polyribosomes and is thought to be involved in the translational efficiency and trafficking of certain mrnas (Jin et al., 2003; Willemsen et al., 2004). FMRP is predominantly a cytoplasmatic protein,

8 266 A Synaptic Model for Pain: Long-Term Potentiation in the Anterior Cingulate Cortex but it does shuttle between the nucleus and cytoplasm, perhaps transporting selective mrna molecules to their final destination within the cell. In neurons, FMRP is found in the dendrite spine head, thereby playing a role in local protein synthesis (Bagni and Greenough, 2005). FMRP was shown to function as a translational repressor for some synaptic proteins, such as Arc, α-camkii, and the dendritic microtubule associated protein 1b (Zalfa et al., 2003). Protein synthesis has been considered a necessary and important component of synaptic morphology and plasticity. The pairing training produced a significant, long-lasting potentiation of synaptic responses in WT mice. However, synaptic potentiation in slices of FMR1 KO mice was completely blocked. This finding provides the first evidence that FMRP may contribute to synaptic potentiation in the ACC neurons (Zhao et al., 2005a). Egr1 The zinc finger transcription factor Egr1 (also called NGFI-A, Krox24, or zif/268) is critical for coupling extracellular signals to changes in cellular gene expression. The upstream promoter region of the Egr1 contains binding sites for cyclic AMP-response elements (CRE), suggesting that Egr1 may act downstream from the CREB pathway. In the ACC neurons, egr1 activity is activated by injury including amputation (Wei et al., 1999). One possible role of Egr1 is to contribute to synaptic potentiation. Genetic deletion of Egr1 mice indeed show defect in ACC LTP, while normal synaptic transmission is observed. Furthermore, NMDA receptor mediated responses; a key component for the induction of cingulate LTP is unaffected. In consistent with synaptic potentiation, behavioral fear and persistent pain are also significantly reduced in Egr1 knockout mice (Ko et al., 2005a; 2005b). Other potential signaling proteins The list of signaling proteins contributing to the ACC is sure to increase in future. Many signaling molecules are reported in the ACC, including nitric oxide (NO), carbon monoxide (CO), cgmp, PKG and BDNF. The failure to demonstrate their contribution to cingulate LTP under experimental conditions do not necessary mean that these molecules are not important for cingulate related higher functions, including those require central plasticity. It is possible that a better cellular model of LTP or neuronal circuit models are yet to be developed/discovered in order to investigate roles of these existing molecules in the ACC. Indeed, there are several reports of in vivo pharmacological evidence for some of these molecules in ACC functions. Expression of cingulate LTP At least four possible mechanisms may contribute to the expression of LTP: (1) presynaptic enhancement of glutamate release; (2) postsynaptic enhancement of glutamate receptor mediated responses; (3) recruitment of previously silent synapses or synaptic trafficking or insertion of AMPA receptors; (4) structural changes. Under in vitro brain slice conditions, it looks like that LTP mechanism may depend on the induction protocol in certain cases. Paired-pulse facilitation (PPF) was not altered after the induction of cingulate LTP (Zhao et al., 2005b). However, we do not rule out the possibility of presynaptic changes in the ACC during other physiological/pathological conditions. Among these possibilities, we have recently investigated the roles of GluR1 and GluR2/3 using genetic and pharmacological approaches. We found that GluR1 subunit C-terminal peptide analog, Pep1-TGL, blocked the induction of cingulate LTP (Toyoda et al., 2007, in press). Thus, in the ACC, the interaction between the C-terminus of GluR1 and PDZ domain proteins is required for the induction of LTP. Synaptic delivery of the GluR1 subunit from extrasynaptic sites is the key mechanism underlying synaptic plasticity (Hayashi et al., 2000; Passafaro et al., 2001) and GluR1-PDZ interactions play a critical intermediate in this plasticity. Our pharmacological experiments show that the application of PhTx 5 min after paired training reduced to synaptic potentiation, while PhTx had no effect on basal responses. Therefore, we believe that Ca 2 -permeable GluR2-lacking receptors contribute to the maintenance of LTP and are necessary for subsequent LTP stabilization. Although our data did not provide direct evidence for the synaptic trafficking or insertion of GluR1 receptors at postsynaptic membrane, the present findings suggest selective contribution of AMPA subtype receptors to cingulate LTP. GluR2/3 subunits may continually replace synaptic GluR2/3 subunits in an activity-independent manner that maintains constant synaptic transmission (Bredt and Nicoll, 2003; Carroll et al., 2001; Malinow and Malenka, 2002; Song and Huganir, 2002). We also examined the role of these peptides in synaptic potentiation in the ACC and found that the GluR2/3-PDZ interaction had no effect on cingulate LTP. We did find that the same interfering peptides inhibited cingulate LTD (Toyoda et al., 2006). These findings suggest that GluR1 and GluR2/3 play different roles in cingulate LTP vs LTD. LTP occurs in the ACC after injury In brain slices, cingulate synapses can undergo LTP after the experimentally designed training protocols. One major question hunting most of in vivo experiments is that the protocol used to induce LTP may be artifact. This has been nicely shown in hippocampus. While LTP mechanisms are well established in both cultured neurons and slices, the roles of LTP in hippocampus-related learning and memory tests remain unclear (Barnes, 1995). A better sample of the link between LTP and behaviors is the fear

9 Min Zhuo 267 memory (Kandel, 2001; Sigurdsson et al., 2007). One reason for studying ACC plasticity is that it is possible linked to the injury-related central plasticity. One important question related to AC plasticity is whether injury causes prolonged or long-term changes in synaptic transmission in the ACC in whole animals. To test this question, we first measure synaptic responses to peripheral electrical shocks. We placed a recording electrode in the ACC of anesthetized rats (Wei and Zhuo, 2001). At high intensities of stimulation, sufficient to activate A δ and C fibers, evoked field EPSPs were found in the ACC. The field EPSPs recorded from the ACC was obviously polysynaptic in nature, likely involving at least primary afferent fibers and spinothalamic and thalamocortical tracts (the estimated latency for the onset of field EPSPs was 12.0 ± 0.1 ms). To detect central plastic changes, we performed amputation at the hindpaw contralateral to the one to which stimulation was delivered. Interestingly, after amputation of a central digit of the hindpaw, we observed a rapid enhancement of sensory responses to peripheral electrical shocks delivered to the normal hindpaw. The potentiation was long-lasting; evoked responses remained enhanced for at least 120 min (Wei and Zhuo, 2001). In order to address whether synaptic changes may occur locally within the ACC, we measured field EPSPs to focal ACC electrical stimulation. We observed a longlasting potentiation of field EPSPs after amputation that lasted for at least 90 min (Wei and Zhuo, 2001). The amount of potentiation is not significantly different from that in field recordings evoked by hindpaw stimulation, suggesting that the ACC is likely due to abnormal activity during and after amputation. One important question is whether potentiated sensory responses required persistent activity from the injured hindpaw. To test this, we locally injected a local anesthetic, QX-314, into the hindpaw (5%, 50 µl) at 120 min after amputation. QX-314 injection did not affect the synaptic potentiation induced by amputation (Wei and Zhuo, 2001). Because it is difficult to determine the origin of electrical changes using field recordings, we performed intracellular recordings from anesthetized rats. We found that there was a long-lasting membrane potential depolarization in ACC neurons of adult rats after digit amputation in vivo (Wu et al., 2005c). Shortly after digit amputation of the hind paw, the membrane potential of intracellularly recorded ACC neurons quickly depolarized from about 70 mv to 15 mv and then slowly repolarized. The duration of this amputation-induced depolarization was about 40 min. Intracellular staining revealed that these neurons were pyramidal neurons in the ACC. A NMDA receptor antagonist MK-801 significantly reduced the depolarization. These results are the direct in vivo electrophysiological evidence that ACC pyramidal cells undergo rapid and prolonged depolarization after digit amputation, and the amputation-induced depolarization in ACC neurons might be associated with the synaptic mechanisms for phantom pain. LTP in the ACC and pathological pain share some common mechanisms While the importance of the ACC in brain cognitive functions is the topic of many future studies, one simple function of ACC LTP may be persistent pain (Zhuo, 2006). Here I will enlist three genetic/pharmacological studies to show that understanding ACC plasticity may indeed help us to control chronic pain in patients. Role of NMDA NR2B overexpression Functional NMDA receptors contain heteromeric combinations of the NR1 subunit plus one or more of NR2A-D (Hollmann and Heinemann, 1994). In humans and rodents, NR2A and NR2B subunits are highly expressed in forebrain structures. NR2A and NR2B subunits confer distinct properties to NMDA receptors; heteromers containing NR1 plus NR2B mediate a current that decays three to four times more slowly than receptors composed of NR1 plus NR2A (Monyer et al., 1994). NMDA receptor mediated currents are long-lasting compared with the rapidly desensitizing kinetics of AMPA and KA receptor channels. Over the developmental stages into the adulthood, the NR2B expression is found to be reduced (Sheng et al., 1994). To examine the function of NR2B, Tang et al. generated transgenic mice with forebrain-targeted NR2B overexpression. In these transgenic mice, the normal developmental change in NMDA receptor kinetics was reversed (Tang et al., 1999). NR2B subunit expression was observed extensively throughout the cerebral cortex, striatum, amygdala, and hippocampus, but not in the thalamus, brainstem, or cerebellum. In both ACC and insular cortex, NR2B expression was significantly increased, and NMDA receptor mediated responses were enhanced (Wei et al., 2001). NMDA receptor mediated responses in the spinal cord, however, were not affected. NR2B transgenic and wild-type mice were indistinguishable in tests of acute nociception, NR2B transgenic mice exhibited enhanced behavioral responses after peripheral injection of formalin. Late phase nociceptive responses but not early responses were enhanced. Furthermore, mechanical allodynia measured in the complete Freund s adjuvant (CFA) model were significantly enhanced in NR2B transgenic mice. These findings provide the first genetic evidence that forebrain NMDA receptors play a critical role in chronic pain. Inhibition of NMDA NR2B receptor Does genetically overexpression of NR2B mimic physiological or pathological conditions? Our recent study provides the first evidence that the up-regulation of NMDA NR2B receptors in the ACC contributing to inflammation-related per-

10 268 A Synaptic Model for Pain: Long-Term Potentiation in the Anterior Cingulate Cortex sistent pain. After persistent inflammation, the expression of NMDA NR2B receptors in the ACC was up-regulated; thereby increasing the NR2B component in NMDA mediated responses (Wu et al., 2005b). Consistently, microinjection into the ACC and systemic administration of NR2B receptor selective antagonists inhibited behavioral responses to peripheral inflammation. These results are in good accordance with our previous report showing that NR2B forebrain overexpression selectively enhanced inflammation-related persistent pain in transgenic mice (Wei et al., 2001). Furthermore, we believe that these findings provide critical evidence that NMDA NR2B receptors undergo long-term plastic changes in the brain after injury. It should be noted that neurons of the ACC have been implicated in other brain functions and our present results do not rule out roles for NMDA NR2B receptors in other ACC-related physiological functions. We believe that this NR2B receptor up-regulation is likely to be reliant on activity-dependent mechanisms. Several lines of evidence support this prediction: (1) The molecular motor protein KIF 17 has been shown to be involved in the active transport of NR2B (Guillaud et al., 2003; Setou et al., 2000; Wong et al., 2002); (2) NR2B, mrna and protein, is highly expressed in ACC neurons (Wei et al., 2001) and (3) NR2B contains a CREB binding domain which may couple increases in intracellular calcium with the increase in NR2B expression. Since NMDA receptors play an important role in activity-dependent plasticity in the ACC, we suggest that NR2B may be regulated through NMDA-calcium-CaM-dependent signaling pathways. The activation of NMDA receptors triggers postsynaptic calcium, leading to the activation of calcium-stimulated CREB in the ACC after injury (Wei et al., 2002b). Since NR2B contains a CREB binding domain, it is likely that NR2B may be activated downstream from the CREB signaling pathway through the activation of NMDA receptors (Fig. 6). A recent study showed that persistent pain induced by tissue inflammation or nerve injury were significantly reduced in PDZ-93 knockout mice, in part due to the lower level of NR2B expression at the spinal and cortical levels of knockout mice (Tao et al., 2003). AC1 and AC8 knockout mice AC1 and AC8, the two major CaM-stimulated ACs in the brain, couple NMDA receptor activation to camp signaling pathways. In the ACC, strong and homogeneous patterns of AC1 and AC8 expression are observed in all ACC layers (Wei et al., 2002b). Behavioral studies found that wild-type, AC1, AC8 or AC1&AC8 double knockout mice were indistinguishable in tests of acute pain including the tail-flick test, hot-plate test, the mechanical withdrawal responses. However, behavioral responses to peripheral injection of two inflammatory stimuli, formalin and CFA, were reduced in AC1 or AC8 single knockout mice. Deletion of Fig. 6. The up-regulation of AMPA and NMDA NR2B receptors in the ACC after peripheral inflammation or nerve injury. Activation of postsynaptic glutamate NMDA receptors leads to an increase in postsynaptic Ca 2 in dendritic spines. Ca 2 binds to CaM and leads to activation of calcium-stimulated ACs, mainly AC1 and other Ca 2 /CaM dependent protein kinases (PKC, CaMKII and CaMKIV). Activation of CaMKIV, a kinase predominantly expressed in the nuclei, will trigger CREB signaling pathways. In addition, activation of AC1 and AC8 lead to activation of PKA, and subsequently CREB as well. MAPK/ ERK could translocate from the cytosol to the nucleus and then regulate CREB activity. Subsequently, postsynaptic synthesis of NMDA NR2B receptor is increased, and together with endogenous motor protein KIF17, these new NR2B subunits are added to postsynaptic NMDA receptors. Such possible positive feedback may further enhance neuronal excitability within the ACC, and contribute to chronic pain. In addition to the up-regulation of NMDA NR2B receptors, it is likely that AMPA receptor will undergo plastic up-regulation. Similar mechanism as described in Fig. 5 may occur during the injury as well. The enhanced AMPA and NMDA receptor mediated responses thus likely to lead to positive enforcement of excitatory transmission within the ACC, and thus contribute to chronic, severe pain as well as pain-related mental disorders. both AC1 and AC8 in AC1&AC8 double knockout mice produced greater reduction in persistent pain (Wei et al., 2002b). More importantly, microinjection of an AC activator, forskolin, can rescue defects in chronic pain in AC1 and AC8 double knockout mice. Consistently, pharmacological intervenes of NMDA receptors as well as camp signaling pathways within the ACC also produced inhibitory effects on persistent pain in normal or wild-type animals, supporting the roles of ACC in persistent pain. Microinjection of NMDA receptor antagonists or campdependent protein kinase (PKA) inhibitors reduced or blocked mechanical allodynia related to inflammation (Wei et al., 2002b). The involvement of AC1 and/or AC8 is also reported in other forms of persistent pain such as chronic muscle pain and neuropathic pain (Vadakkan et al., 2006). Summary and future directions It is clear now that neuronal synapses in the ACC, a critical area for processing sensory perception, cognition, ex-

11 Min Zhuo 269 ecutive function, emotion, can rapidly undergo long-term plastic changes. Thus, each new experience is likely to trigger changes in these highly plastic neuronal synapses, and subsequently, altered or enhanced synapses affect experience to future new stimuli. It is also true that such plasticity may serve as pathological basis for many ACC related mental disorders. The secret of ACC is just starting to be revealed. Here I would like to discuss three major possible research questions that I believe is highly relevant for ACC plasticity. First, are LTP equally occurring in the thalamic inputs and cortical-cortical connections? In brain slice preparation, it is difficult to stimulate selectively thalamic inputs. Focal stimulation in the previous studies may stimulate thalamic inputs, cortical inputs from the opposite ACC or other cortical areas. Previous studies in brain slices as well as preliminary results in vivo show that ACC-ACC synapses and thalamus-acc synapses may both under LTP after training or injury (Wu et al., 2005b, Zhuo, unpublished data). It will be critical in future to use in vivo approaches, and/or in vitro preparation if possible, to investigate if LTP in these two different pathways onto the cingulate neurons may share similar mechanisms. More importantly, if one affects the other, both during the phase of the induction and expression. Second, are there any presynaptic contributions to the ACC LTP? We recently report that an enhancement of synaptic transmission in the ACC of animals suffering from chronic inflammatory pain (Xu et al., unpublished data; Zhao et al., 2006). This is demonstrated by the increased input-output curves and decreased paired-pulsed facilitation in ACC slice recordings. Furthermore, we show that the enhancement in synaptic transmission is the result of an increased presynaptic probability of neurotransmitter release in ACC synapses, as demonstrated by the increased mepsc frequency as well as faster blocking rate of NMDA EPSCs by MK-801. In addition, analysis of the I V relationship and reversal potential of AMPA receptor mediated mepscs showed no difference between control and CFA-injected mice. Taken together, these results suggest that the enhanced synaptic transmission results from the increased probability of presynaptic neurotransmitter release rather than a possible postsynaptic modification of functional AMPA receptors. However, we also found that NMDA receptor dependent ACC LTP is purely postsynaptic expressed (Zhao et al., 2005b). Thus it is likely that different forms of LTP may exist in the ACC. New form of LTP containing presynaptic component may remain to be discovered. Future experiments are clearly needed to investigate these possibilities. Finally, what is the impact of synaptic LTP on neuronal coding within the ACC? One key question for synaptic plasticity is the impact of LTP on neuronal action potentials. Is LTP contributing to increases in neuronal firing to subsequent stimuli such as in case of hyperalgesia and allodynia? Or LTP contribute to special activity within a neuronal circuit? In summary, integrative approaches have yielded significant progresses in our understanding of synaptic mechanisms for sensory transmission, modulation and plasticity in the ACC. The involvement of each signaling protein will be identified. The combination of different experimental approaches such as synaptic neurobiology, system biology and neuronal circuits is clearly needed to understand how synaptic plasticity affects human and animal behaviors under physiological and pathological conditions. Acknowledgment I thank funding supports from the EJLB- CIHR Michael Smith Chair in Neurosciences and Mental Health in Canada, CIHR operating grants and NIH NINDS References Bagni, C. and Greenough, W. T. (2005) From mrnp trafficking to spine dysmorphogenesis: the roots of fragile X syndrome. Nat. Rev. Neurosci. 6, Barnes, C. A. (1995) Involvement of LTP in memory: are we searching under the street light? Neuron 15, Bliss, T. V. and Collingridge, G. L. (1993) A synaptic model of memory: long-term potentiation in the hippocampus. Nature 361, Botvinick, M. M., Cohen, J. D., and Carter, C. S. (2004) Conflict monitoring and anterior cingulate cortex: an update. Trends Cogn. Sci. 8, Bredt, D. S. and Nicoll, R. A. (2003) AMPA receptor trafficking at excitatory synapses. Neuron 40, Calejesan, A. A., Kim, S. J., and Zhuo, M. (2000) Descending facilitatory modulation of a behavioral nociceptive response by stimulation in the adult rat anterior cingulate cortex. Eur. J. Pain. 4, Carroll, R. 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