Central Sensitization, Synaptic Potentiation, and Microglia

FIGURE 1 Long-term potentiation (LTP) as a cellular model for chronic pain in the anterior cingulate cortex (ACC). (A) Activation of immediate early genes in ACC neurons of an adult rat after peripheral injury. (B) In vivo recording of ACC LTP in adult rats after amputation of a single digit in the hind paw under anesthesia. (C) Current-clamp recordings to identify pyramidal neurons (i) and interneurons (ii) of adult mice by current injections of −100, 0 and 100 pA. A labeled pyramid-like neuron is shown in (iii). RP: resting membrane potential. (D) LTP was induced in pyramidal neurons in adult mouse ACC by the pairing training protocol (indicated by an arrow). The insets show averages of six excitatory postsynaptic currents (EPSCs) 5 min before and 25 min after the pairing training (arrow). The broken line indicates the mean basal synaptic responses. (E) Field recording of late-phase LTP (L-LTP) in adult mouse ACC slices. (Adapted from Zhuo M. Targeting neuronal adenylyl cyclase for the treatment of chronic pain. Drug Discov Today 2012;17:573–82).

Heterosynaptic LTP

In addition to homosynaptic LTP, heterosynaptic LTP has been also reported. 5-hydroxytryptamine (5-HT), an important neurotransmitter of the raphe-spinal projection pathway, transforms silent glutamatergic synapses into functional ones [9,11,20]. The mechanism underlying this conversion involves 5-HT induced PKC activation, AMPA receptor-PDZ interactions, and the recruitment of AMPA receptors. Silent synapses are likely involved in synaptic potentiation in the spinal dorsal horn, considering that the recruitment of silent synapses could significantly enhance spinal sensory transmission, including nociceptive transmission. Another potential function of silent synapses is to contribute to a descending facilitatory modulatory network within the spinal cord [38]. The recruitment by 5-HT could strengthen spinal sensory synapses receiving innervation from descending 5-HT projection fibers, which most likely originate from the rostral ventromedial medulla (RVM) [39].

LTP IN PAIN-RELATED CORTEX

ACC and IC

Human and animal studies are consistent with the suggestion that the ACC, IC and related areas are important for pain perception [7,19,40]. Both ACC and IC neurons respond to nociceptive stimuli and activity within the ACC is related to the unpleasantness or discomfort of noxious stimuli. Peripheral injury caused bilateral increases of activity-dependent immediate early genes such as c-fos, Egr1 and CREB, and increased electrophysiological responses. Electrophysiological experiments in cortical slices have shown that excitatory synaptic transmission in the ACC and IC is primarily glutamatergic [40].

It is commonly thought that mature cortical synapses are less plastic. In certain cortical areas, LTP is found to be age-dependent under experimental conditions. The disappearance of cortical LTP is related to the disappearance of glutamate silent synapses or maturation of cortical synapses. However, we found that the ability of cortical synapses to undergo LTP is mostly dependent of induction protocol. In both ACC and IC synapses, theta burst stimulation (TBS), can induce LTP in synapses of young and adult animals (Fig. 1). Peripheral noxious foot shocks induced TBS-like neuronal activities of ACC neurons by in vivo recordings of freely moving mice [17]. In addition, LTP also can be induced using two other protocols, including the pairing training protocol and the spike-EPSP pairing protocol [37].

Pharmacological studied using selective receptor antagonists reveal that ACC LTP exists in at least four different forms: NMDA receptor dependent, L-type voltage-gated calcium channel (L-VGCC) dependent, late-phase LTP (L-LTP) and presynaptic LTP (pre-LTP) under experimental in vitro brain slice conditions.

NMDA Receptor Dependent LTP

In ACC synapses, LTP induced by different protocols are sensitive to the inhibition of NMDA receptors [37,40]. Application of a NMDA receptor antagonist AP-5 blocked the induction of LTP. NMDA receptor containing GluN2A (or called NR2A) or GluN2B (NR2B) subunits contribute to most of NMDA receptor currents, application of a GluN2A antagonist NVP-AAM077 and GluN2B antagonist ifenprodil or Ro 25–6981 produce an almost completely blockade of NMDA receptor mediated EPSCs. Application of GluN2A or GluN2B antagonist also reduces LTP, without complete abolishment of LTP. LTP only is abolished after the co-application of both inhibitors.

Calcium Channel Dependent LTP

L-VGCCs are also required for the induction of LTP by TBS in the field recording condition [13]. Unlike LTP recorded using field recording, LTP recorded using whole-cell patch clamp does not respond to the inhibition of L-VGCCs [37].

Protein Synthesis Dependent L-LTP

Recent studies using a 64-channel multi-electrode array (MED64) show that ACC LTP induced by multiple TBS can last more than 5 hours. This form of potentiation is sensitive to inhibition of protein synthesis [3,14], indicating that protein synthesis dependent L-LTP exists in the ACC. It is likely that L-LTP may contribute to long-term changes in the cortical circuits that are triggered by peripheral injury. Future investigations of basic mechanisms are clearly needed for ACC L-LTP.

MOLECULAR MECHANISM FOR CORTICAL LTP

Recent genetic, pharmacological and electrophysiological approaches have been used to investigate the basic mechanisms for LTP in the ACC synapses [21,40,41]. Activation of NMDA receptors leads to an increase in postsynaptic Ca2+ in dendritic spines. Ca2+ serves as an important intracellular signal for triggering a series of biochemical events that contribute to the expression of LTP. Ca2+ binds to calmodulin (CaM) and leads to activation of calcium-stimulated signaling pathways. Furthermore, postsynaptic injection of BAPTA completely blocked the induction of LTP, indicating the importance of elevated postsynaptic Ca2+ concentrations. A work using electroporation of mutant CaM in the ACC neurons suggest that calcium binding sites of CaM are critical for the induction of ACC LTP. Ca2+-stimulated, neuron-specific adenylyl cyclase subtype 1 (AC1) is highly expressed in the ACC neuron and LTP induced by TBS or pairing stimulation are abolished in AC1 knockout mice. Several other signaling proteins or protein kinases are found to be involved in ACC LTP, including Ca2+-CaM-dependent protein kinase IV (CaMKIV), early growth response gene 1 (egr1), mitogen-activated protein kinase (MAP kinase) and fragile X mental retardation protein (FMRP).

At least four different synaptic mechanisms may contribute to the expression of LTP: (1) Presynaptic enhancement of the release of glutamate; (2) Postsynaptic enhancement of glutamate AMPA receptor mediated responses; (3) Recruitment of previously ‘silent’ synapses or synaptic trafficking or insertion of AMPA receptors; and (4) Structural changes in synapses. 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 ACC LTP [40]. 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 and GluR1-PDZ interactions play a critical role in this type of plasticity. Application of Philanthotoxin-433 (PhTx) 5 min after LTP induction reduced synaptic potentiation, while PhTx had no effect on basal AMPA receptor-mediated responses, suggesting that Ca2+-permeable GluR2-lacking receptors contribute to the maintenance of ACC LTP. Our recent studies found that ACC LTP is absent GluR1 knockout mice [18]. We also examined the role of GluR2 related peptides in synaptic potentiation in the ACC and found that the GluR2/3-PDZ interaction had no effect on ACC LTP, and the same interfering peptides inhibited ACC LTD.

Intracellular Signaling Pathways for Required for Synaptic Potentiation

Ca-CaM

Activation of glutamate NMDA receptors leads to an increase in postsynaptic Ca2+ in dendritic spines. Ca2+ serves as an important intracellular signal for triggering a series of biochemical events that contribute to the expression of LTP. Ca2+ binds to CaM and leads to activation of calcium-stimulated signaling pathways [27]. Furthermore, postsynaptic injection of BAPTA completely blocked the induction of LTP, indicating the importance of elevated postsynaptic Ca2+ concentrations [37]. 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 [27].

Adenylyl Cyclases: AC1, AC8

cAMP signaling pathways that are the signaling pathways in biological systems. Among more than 10 subunits, AC1 and AC8 are two AC subtypes that respond positively to calcium-CaM, including in pain-related spinal and cortical neurons [40,41]. 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 [25]. 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 [13]. AC1 is also contributing to synaptic potentiation induced by forskolin, an AC activator that is non-selective for AC isoform. Gene deletion of AC8 subunit partially contributes to forskolin-induced 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 [22].

CaMKIV

A major neuronal signaling pathway by which Ca2+ activates CREB involves the Ca2+/calmodulin dependent protein kinase type IV (CaMKIV). CaMKIV is a member of a group of multifunctional CaMK, so called because of their broad substrate specificity, which also includes CaMKI and CaMKII. 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 [24]. 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 Ca2+-CaM complexes by nuclear CaM binding proteins. The abolishment of CaM translocation in CaMKIV knockout mice identifies CaMKIV as the critical sink that traps Ca2+-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 CaMKIV 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 [24].

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 10–30 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. FMRP is predominantly a cytoplasmatic protein, 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. 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. 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 [36].

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 [23]. 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, and persistent pain are also significantly reduced in Egr1 knockout mice [5,6].

Based on progress made as described above, a proposed synaptic model for the molecular mechanism of LTP in the ACC based on these studies is shown in Figure 2

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