Cell Tissue Res (2014) 357:407–426 DOI 10.1007/s00441-014-1942-5

REVIEW

Neuronal calcium signaling in chronic pain Anna M. Hagenston & Manuela Simonetti

Received: 16 April 2014 / Accepted: 3 June 2014 / Published online: 12 July 2014 # Springer-Verlag Berlin Heidelberg 2014

Abstract Acute physiological pain, the unpleasant sensory response to a noxious stimulus, is essential for animals and humans to avoid potential injury. Pathological pain that persists after the original insult or injury has subsided, however, not only results in individual suffering but also imposes a significant cost on society. Improving treatments for longlasting pathological pain requires a comprehensive understanding of the biological mechanisms underlying pain perception and the development of pain chronicity. In this review, we aim to highlight some of the major findings related to the involvement of neuronal calcium signaling in the processes that mediate chronic pain. Keywords Sensitization . Chronic pain . Pain pathways . Calcium signaling . Nuclear calcium

Introduction The sensation of pain is important for the survival of the individual. Pain sensation is propagated from the periphery into the central nervous system via pain pathways originating in primary sensory neurons whose cell bodies are situated within the dorsal root ganglia (DRG). These neurons have terminal endings within the skin and viscera, where noxious stimuli from the environment are detected, and carry this information via unmyelinated C fibers and myelinated Aδ fibers to the spinal cord dorsal horn, where they form A. M. Hagenston (*) University of Heidelberg, Neurobiology, Im Neuenheimer Feld 364, 69120 Heidelberg, Germany e-mail: [email protected] M. Simonetti University of Heidelberg, Pharmacology, Im Neuenheimer Feld 364, 69120 Heidelberg, Germany

excitatory synapses with neurons in laminae I, II, and V. Second order neurons in the spinal cord dorsal horn project in turn via the spinothalamic and spinoreticulothalamic relays to the somatosensory and limbic systems of the cortex, respectively, where the nociceptive and emotional/aversive components of pain are processed (Millan 1999). Together with these ascending pain pathways that convey pain stimuli from the periphery to higher brain centers, there are also painmodulating descending pathways. These arise in the brainstem, hypothalamus, and cortical structures, and modulate both sensory inputs from primary afferent fibers and projection neurons in the dorsal horn of the spinal cord (Millan 1999, 2002). The best known of these pathways are the axes of the periaqueductal gray and rostroventral medulla, which can both inhibit or facilitate sensory processing in the spinal cord dorsal horn. Just as the sensation of pain involves the activity of a neuronal network spanning from the periphery to the spinal cord to the brain and back, so too the development of pain hypersensitivity involves changes at all levels of this network, beginning with heightened sensitivity of peripheral neurons (peripheral sensitization), altered pain processing within the spinal cord and deriving from descending projections (central sensitization), and finally via changes in the experience of pain following from altered cerebral network activity (also central sensitization). Sensitization of the neuronal networks underlying pain perception results behaviorally in spontaneously occurring pain, increased responsiveness to noxious stimuli (hyperalgesia), and nociceptive responsiveness to otherwise innocuous stimuli (allodynia). In the periphery, this sensitization is characterized by an increase in the basal firing rates of primary sensory neurons, their elevated responsiveness to noxious stimuli, and a decrease in their activation thresholds for thermal and mechanical stimulation (Woolf and Ma 2007). Central sensitization at the level of the spinal cord is considered to result from activity-dependent changes in spinal neuronal function, and involves both the long-term

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potentiation (LTP) of individual synapses from peripheral afferents onto spinal neurons as well as the increased excitability of neurons within the spinal cord dorsal horn (Ji et al. 2003; Sandkühler 2009). Within the somatosensory and limbic systems of the brain, central sensitization takes the form of heightened neuronal responsiveness and neural circuit reorganization (Moseley and Flor 2012). The neuronal plasticity associated with peripheral and central sensitization manifests itself in a range of different functional and structural plastic changes that together lead to longlasting alterations in peripheral sensitivity and spinal and cortical neuronal networks, an elevated net spinal output, an altered perception of pain, and the development of a pathological pain state. Functionally, neuronal plasticity can occur on at least four levels, including (1) changes in neuronal excitability and signal transduction resulting from altered protein expression of and/or post-translational modifications to existent proteins; (2) changes in synaptic strength deriving postsynaptically from changes in the number and/or synaptic localization of neurotransmitter receptors and/or presynaptically from changes in neurotransmitter release probability; (3) changes in synaptic connectivity resulting from modifications in the density and/or size of synaptic spines, altered complexity of neuronal dendritic arbors and/or the numbers of axonal afferents; and (4) changes in the numbers of cells that collectively respond to a given noxious stimulus. Our aim here is to highlight the role of calcium signaling in some of these neuronal plastic processes. Numerous diseases of the central nervous system, including pathological pain, are associated with abnormal neuronal calcium homeostasis and calcium signaling (Fernyhough and Calcutt 2010; Mattson 2012; Stutzmann 2007). Indeed, aberrant calcium channel physiology and expression has been implicated in not only neuropathic pain and diabetic neuropathy but also chronic inflammatory pain (Bourinet et al. 2014; Fernyhough and Calcutt 2010; Naziroglu et al. 2012). Nevertheless, a number of marked differences in the underlying mechanisms of the different pain modalities have been identified, and results from studies examining calcium regulation in a variety of different pain models suggest the existence of specific patterns of injury and/or disease-induced changes in the regulation of calcium (Xu and Yaksh 2011). Regardless of the particular mechanisms involved, the net output in all of the different forms of chronic pain is an over-activation of pain-transducing primary sensory neurons in the DRG and an enhanced excitation of second order neurons in the spinal cord dorsal horn.

Neuropathic pain Neuropathic pain is defined as pain whose initiating cause is a primary lesion or dysfunction in the nervous system. In the

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spinal cord, neuropathic pain is characterized by the increased responsiveness and spontaneous activity of dorsal horn neurons, the expansion of the receptive fields of these neurons, and a loss of GABAergic inhibition (Campbell and Meyer 2006; Kuner 2010; von Hehn et al. 2012). Many of the mechanisms proposed to explain the primary genesis of neuropathic pain, however, involve the increased excitability or enhanced spontaneous activity of primary afferents. Such elevated activity may result from a number of possible functional alterations in the afferent cells, including the increased sensitivity and/or activity of peripheral terminals, cell bodies, and afferent axons of injured neurons, the formation by injured cells of ectopic neuronal pacemakers along sensory nerve fibers, and the increased electrical excitability of adjacent, uninjured axons (Campbell and Meyer 2006; von Hehn et al. 2012). It is well established, for instance, that nerve injury in general and axotomy in particular leads to increased neuronal excitability of DRG neurons (Study and Kral 1996; Wall and Devor 1983). This increased excitability is strongly associated with decreases in voltage-dependent calcium currents, a decrease in resting calcium concentrations in injured DRG neurons, a decrease in the magnitude of evoked calcium transients specifically in injured putative nociceptive DRG neurons, and finally a diminished recruitment of calciumsensitive potassium channels (Fuchs et al. 2005, 2007; Hogan 2007).

Diabetic neuropathy Diabetic neuropathy is one of the most common peripheral neuropathies. In patients, diabetic neuropathy leads to the degeneration of peripheral nerve fibers and a concomitant loss of sensory perception. One of the foremost symptoms of diabetic neuropathy is pain. Both the painful and degenerative aspects of diabetic neuropathy have been linked to abnormalities in calcium signaling and homeostasis. Indeed, disturbed calcium homeostasis has been observed in a variety of tissues from animals with experimentally-induced diabetes as well as from diabetic patients, and in sensory neurons typically consists of an increase in the resting intracellular calcium concentration, the decreased activity of calcium transporters, and a reduction in stimulus-triggered calcium rises (Fernyhough and Calcutt 2010; Verkhratsky and Fernyhough 2008). One source of these changes is a decrease in the rate of calcium extrusion following stimulation in diabetic neurons, which can result from a decrease in the expression of plasma membrane calcium pumps as well as a reduction in the calcium uptake by the neuronal endoplasmic reticulum (ER) and mitochondria (Fernyhough and Calcutt 2010; Verkhratsky and Fernyhough 2008; Zherebitskaya et al. 2012). Another major source of calcium dysregulation in diabetic neuropathy is a change in the entry of calcium through plasma membrane

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voltage-dependent calcium channels (VDCCs) (Todorovic and Jevtovic-Todorovic 2014). In rodent models of diabetes, for example, enhanced mRNA levels of and currents from Ttype VDCCs have been reported in putative nociceptive DRG neurons, and these changes are correlated with the development of diabetic pain (Hall et al. 1995; Jagodic et al. 2007; Khomula et al. 2013; Yusaf et al. 2001). Moreover, both the pharmacological inhibition and knockdown of T-type channels was able to reduce pain in animal models of diabetes (Latham et al. 2009; Messinger et al. 2009; Obradovic et al. 2014). These results, and others, suggest that increased expression of T-type VDCC channels may contribute to the underlying mechanisms of painful diabetic neuropathy (Todorovic and Jevtovic-Todorovic 2014). A particularly interesting molecular mechanism for controlling the expression and function of T-type VDCC subunits under hyperglycemic conditions—as independently shown in two recent papers—involves their asparagine-linked glycosylation (Orestes et al. 2013; Weiss et al. 2013). Despite some differences, possibly due to different cell culture conditions, together these studies convincingly demonstrate that pathological glucose levels in a recombinant cell culture system in vitro and in the leptin-deficient mouse model of peripheral diabetic neuropathy in vivo lead to the glycosylation of extracellular asparagine residues of CaV3.2 subunits, and that this glycosylation results in the augmented surface expression, increased current density, and accelerated current kinetics of T-type VDCCs (Orestes et al. 2013; Weiss et al. 2013). These findings raise the exciting possibility that pharmacological agents directed at glycosylation sites on T-type VDCCs may provide a significant therapeutic benefit to sufferers of painful diabetic neuropathy. Indeed, peripheral application of a glycoside hydrolase both inhibited native T-type currents and reversed hyperalgesic responses in leptin-deficient diabetic mice (Orestes et al. 2013). Although these two studies focused their efforts on the examination of CaV3.2, the most prevalent T-type VDCC isoform in small DRG cells, all three isoforms of T-type VDCCs have been observed in DRG neurons (Choi et al. 2007; Talley et al. 1999). Given the high degree of conservation of asparagine glycosylation sites between Ttype VDCC isoforms, it is therefore conceivable that glycosylation may also modulate the expression and function of CaV3.1 and CaV3.3 T-type VDCC isoforms (Orestes et al. 2013; Weiss et al. 2013). In light of this, it is interesting to note that at least two recent studies have suggested that the modulation of T-type VDCC-mediated currents in DRG neurons may involve changes not only in the surface expression but also in the isoform composition of these channels (Khomula et al. 2014; Yue et al. 2013). In particular, specific types of diabetic neuropathy were associated with the differential remodeling of T-type VDCC isoforms which have distinct biophysical properties, such that the development of thermal hyperalgesia

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specifically (versus hypo- or normalgesia) involved an increase in the peak current density associated with fast inactivating T-type current and a lack of slowly inactivating T-type currents in a subset of nonmyelinated DRG neurons (Khomula et al. 2014). While fast inactivating T-type currents were mediated predominantly by CaV3.2 subunits, slow inactivating T-type currents were mediated by CaV3.2 and other T-type VDCC subunits. The lack of slow T-type currents in diabetic thermal hyperalgesia thus suggests an elimination either of neurons expressing this current or of the underlying subunits (Weiss et al. 2013). Chronic inflammatory pain When peripheral tissues are chronically inflamed, primary sensory nerve terminals are surrounded by a diversity of chemical mediators that stimulate and sensitize DRG neurons, resulting in a number of long-lasting functional changes (Basbaum et al. 2009). Two prominent changes in DRG neurons in models of chronic inflammatory pain are increases in the basal concentration of cytosolic calcium ions and altered voltage-dependent calcium currents (Lu and Gold 2008; Lu et al. 2010; Waxman and Zamponi 2014). Resting calcium levels in medium diameter sensory neurons, for example, were seen to be higher, and depolarization-evoked calcium transients to be larger and to decay more slowly in small and medium diameter sensory neurons from inflamed tissues (Lu and Gold 2008). Consistent with an increase in the expression and/or permeability of plasma membrane ion channels, the altered calcium regulation of sensory neurons has been observed to be accompanied by a notable elevation of their excitability and resting membrane conductance (Dang et al. 2005; Flake et al. 2005; Moore et al. 2002). Indeed, the mechanisms underlying peripheral and central sensitization during inflammation involve the increased surface trafficking of sodium- and calcium-permeable inflammatory receptors and voltage-gated ion channels in primary sensory neurons (e.g., ASIC1, TRPV1, TRPA1, P2X3, NaV1.8, and N-type VDCCs), but also changes in the trafficking and composition of synaptic and extrasynaptic glutamate receptors (reviewed in Bourinet et al. 2014; Ma and Quirion 2014; see below). Interestingly, however, the increased excitability of sensory neurons during inflammation seems not to involve upregulated calcium influx at the soma, but is thought instead to specifically involve changes in calcium signaling within sensory terminals and presynaptic endings (Basbaum et al. 2009; Katz and Gold 2006). Accordingly, the contribution of increased calcium levels in DRG cells to inflammatory hyperalgesia is most likely to result from the facilitation of transmitter release both in the periphery and at central terminals, and the consequent enhancement of both neurogenic inflammation and nociceptive neurotransmission, respectively.

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Calcium sources and sinks Calcium is an integral second messenger in neurons, and is involved in the functional and structural neuronal plasticity that underlies peripheral and central sensitization at all levels. Changes in the intracellular concentration of calcium ions in peripheral and central neurons are mediated by calcium entry through ligand-gated channels such as N-methyl-D-aspartate receptors (NMDARs), calcium-permeable α-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate receptors (AMPARs), and ATP-responsive purinoreceptors (P2XRs) that respond to synaptically released neurotransmitter and glial- or microglial-derived ATP, respectively; through acidsensing ion channels (ASICs), which are activated by extracellular protons; through transient receptor potential (TRP) channels such as the heat- and capsaicin-responsive vallinoid (TRPV) receptors; through VDCCs, including both highvoltage-activated (HVA) and low-voltage-activated (LVA) subtypes that are activated in response to neuronal depolarization (see Bourinet et al. 2014 for a comprehensive overview of NMDARs, P2XRs, TRPs, and ASICs in afferent pain pathways and their roles in pain pathophysiology). Changes in the concentration of calcium ions in neurons along pain pathways are also mediated by the release of calcium from intracellular stores following activation of G-protein-coupled receptors (GPCRs) or receptor tyrosine kinases (RTKs) by their respective ligands and subsequent stimulation of ER inositol trisphosphate receptors (IP3Rs); by the transport out of the mitochondrial lumen via sodium/calcium/lithium exchangers (NCLX); and by the intracellular release of calcium following the calcium-dependent activation of ER ryanodine receptors (RyRs). Although calcium sources typically represent the more popular subjects for study, calcium sinks and extrusion mechanisms, too, are important modulators of calcium signaling, and have also been implicated in the neuronal mechanisms underlying pain sensitization. Neuronal calcium sinks include plasma membrane calcium ATPases (PMCA) and sodium/calcium exchangers (NCXs), which remove cytosolic calcium into the extracellular space; sarco/endoplasmic reticulum calcium ATPases (SERCA pumps), which pump calcium ions into the ER; nuclear NCXs, which remove nuclear calcium into the nucleoplasmic reticulum; the mitochondrial uniporter (MCU), which mediates mitochondrial calcium uptake; and a wide range of calcium binding proteins (CaBPs), including both calcium buffers like calbindin D28k and signaling molecules such as calmodulin (CaM) (Fig. 1).

NMDARs and AMPARs The activation of NMDARs and AMPARs is considered to be a key trigger for activity-dependent changes in neuronal

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excitability and function. NMDARs and AMPARs are expressed at excitatory postsynaptic sites throughout the spinal cord and supraspinal central nervous system, but also in presynaptic primary sensory neurons and nerve terminals (Millan 1999; Polgar et al. 2008; Wu et al. 2004). Consistent with their involvement in the mechanisms underlying peripheral and central sensitization, the altered expression and/or function of both AMPARs and NMDARs has been observed in altered pain states in primary sensory neurons, in the spinal cord dorsal horn, and in supraspinal regions important for pain sensation and pain perception (Basbaum et al. 2009; Ho et al. 2013; Liu and Salter 2010; Neugebauer et al. 2009; Zhou et al. 2014; Zhuo et al. 2011; and others). NMDARs draw particular attention in the context of neuronal plasticity and pain sensitization due to two key features. First, on account of their calcium permeability, NMDARs represent a major source of synaptic activity-dependent calcium rises. Indeed, following their involvement in regulating a broad range of kinases, phosphatases, and other enzymes, NMDAR-mediated calcium rises have been implicated in nearly all aspects of pain plasticity (Woolf and Salter 2000; Wu and Zhuo 2009; Zhuo et al. 2011), and NMDAR antagonists represent an attractive target in chronic pain therapy (Chizh and Headley 2005; Fisher et al. 2000; Wu and Zhuo 2009). Second, the combined sensitivity of NMDARs to both synaptically released neurotransmitter and membrane potential via the voltage-dependent blockade of the ion-conducting pore by extracellular magnesium ions endows them with the unique ability to act as coincidence detectors for simultaneous presynaptic glutamate release and postsynaptic action potential generation. Indeed, modulation of extracellular magnesium concentrations—and thereby of NMDAR activity and NMDAR-mediated calcium influx—has been identified both as a potential source of pain hypersensitivity and as a potential target for pain therapy (Begon et al. 2000; Felsby et al. 1996; Rondon et al. 2010). Although NMDARs likely represent the most recognized postsynaptic source of neuronal calcium entry in neurons, AMPARs, depending on their subunit composition, can also contribute to postsynaptic calcium rises and calciumdependent plasticity. In particular, AMPARs lacking the GluA2 (formerly called GluR2) subunit are conferred with a much increased calcium permeability and modified current rectification and total channel conductance (Hollmann et al. 1991). A number of lines of evidence implicate AMPARs in general, and calcium-permeable AMPARs in particular, in the signaling mechanisms underlying pain chronicity. It has been shown, for instance, that peripherally located AMPARs are at least partially responsible for the transmission of acute nociceptive stimuli from muscle fibers and for the development of irritant-induced nocifensive behaviors (Chun et al. 2008). Similarly, the deletion of calcium-permeable AMPARs from peripheral, pain-sensing neurons results in decreased hypersensitivity and sensitization in models of chronic

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Fig. 1 Calcium sources and sinks. Calcium can enter the cytosol of neurons along the pain axis from the extracellular space via NMDARs and calcium-permeable AMPARs, ATP-responsive P2XRs, ASICs, TRP channels, and VDCCs. Calcium can also enter the cytosol when it is released from intracellular calcium stores via RyRs, or via IP3Rs following the activation of Gq/11-coupled GPCRs or RTKs on the plasma membrane. Calcium can enter the cytosol from the mitochondrial matrix via NCLX, and can be taken up into mitochondria via the MCU. Calcium can also be removed from the cytosol when it is taken up into the ER via SERCA pumps and nuclear NCX, bound by cytosolic CaBPs, or extruded into the extracellular space via PMCAs or NCXs. Cytosolic calcium can freely diffuse through nuclear pores into the nucleus, where it regulates the activity of a wide range of transcription factors (TFs) and controls the expression of diverse target genes

inflammatory pain and arthritis (Gangadharan et al. 2011). Further, the specific antagonism of GluA2-lacking AMPARs was found to alleviate inflammatory pain without affecting animal responses to physiological painful stimuli, indicating an important pathophysiological role for peripheral calciumpermeable AMPARs in chronic pain states (Gangadharan et al. 2011). Indeed, while mice lacking calcium-permeable AMPARs exhibit both a loss of nociceptive plasticity in vitro and a reduction in inflammatory hyperalgesia in vivo, an increase in calcium-permeable AMPARs in GluA2-deficient mice facilitates spinal cord LTP, nociceptive plasticity, and inflammatory hyperalgesia (Hartmann et al. 2004; Youn et al. 2008). Moreover, mice that do not possess calcium-permeable AMPARs lose potentiation and spatial spread of spinal calcium transients induced by peripheral inflammation, indicating that spinal AMPARs are crucial for activity-dependent changes in synaptic processing of nociceptive inputs (Luo et al. 2008). Interestingly, neurons of the spinal cord dorsal horn not only express an unusually high density of calciumpermeable AMPARs (Gu et al. 1996; Tong and MacDermott 2006) but their expression levels relative to calciumimpermeable AMPARs are increased in animal models of both persistent inflammatory and neuropathic pain (Chen et al. 2013; Park et al. 2009; Vikman et al. 2008). These expression changes depend on NMDAR activation, the calcium-dependent internalization of GluA2 AMPAR

subunits, and the insertion of GluR1 AMPAR subunits (Chen et al. 2013; Larsson and Broman 2008; Park et al. 2009). Importantly, increased expression of calciumpermeable AMPARs at postsynaptic sites, for example following the calcium/CaM kinase II (CaMKII)-dependent recruitment of the AMPAR-interacting protein GRIP or the activation of protein kinase C (PKC), both enhances postsynaptic excitation and postsynaptic calcium influx and strengthens the AMPAR-mediated component of spinal synaptic transmission (Galan et al. 2004; Gu et al. 1996; Hartmann et al. 2004; Kopach et al. 2013; Park et al. 2009). Calcium entry through AMPARs may also contribute to the down-regulation of inhibitory glycinergic synaptic function in spinal cord neurons via a mechanism involving CaMKII and calcineurin (Xu et al. 1999). Finally, GluR1-, but not GluA2containing AMPARs in the anterior cingulate cortex, a cortical brain region that, together with the somatosensory cortex and amygdala, plays an important role in pain perception and central sensitization, are implicated in the stimulation of MAP kinases and the induction of LTP in inflammatory pain models in vivo, and their expression levels are elevated in a rat model of visceral hypersensitivity (Toyoda et al. 2009; Zhou et al. 2014). Importantly, the excessive activation of glutamate receptors in general, and NMDARs in particular, is strongly implicated in the mechanisms underlying excitotoxicity in neurons, and

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cell death has been reported in the central nervous system in models of neuropathic pain (Hardingham 2009; Hardingham and Bading 2010; Leong et al. 2011). It is therefore particularly interesting that, in models of peripheral inflammation and neuropathic pain, which lead to increased spontaneous and evoked activity of spinal nociceptive afferents, neurons may express a mechanism by which NMDARs can regulate their own activity in a negative feedback loop (Tappe et al. 2006). A principal player in this feedback loop is Homer1, an adapter protein that in neurons is concentrated in dendritic spines and at the postsynaptic density (Jardin et al. 2013; Xiao et al. 2000). Interestingly, whereas the Homer1b and Homer1c variants link G-protein-coupled metabotropic glutamate receptors (mGluRs) to internal calcium release channels (IP3Rs) at the ER, and to VDCCs and components of the NMDAR complex at the cell surface, the short, activitydependent splice variant Homer1a antagonizes these interactions (Ango et al. 2001; Jardin et al. 2013; Sala et al. 2003). Consistent with the possibility that Homer1a may regulate synaptic NMDAR and AMPAR activity, cells in the hippocampus of Homer1a knockout animals exhibited larger AMPA/NMDA current ratios than wild-type animals, and a redistribution of GluA2 subunits from the dendritic compartment to the soma. Cells in the hippocampus of transgenic Homer1a over-expressing animals, in contrast, exhibited smaller postsynaptic AMPA/NMDA current ratios, an increase in the proportion of GluA2-containing, calciumimpermeable AMPARs at the synapse, and an abolishment of the maintenance phase of LTP (Rozov et al. 2012). In the spinal cord dorsal horn, up-regulation of Homer1a was observed following chronic constriction injury and peripheral inflammation, and this up-regulation depended on the activation of NMDARs and the subsequent stimulation of the MAP kinase pathway (Miyabe et al. 2006; Tappe et al. 2006). Whereas prevention of Homer1a up-regulation using shRNA exacerbated the inflammatory pain phenotype, viral-mediated over-expression of Homer1a attenuated inflammatory hypersensitivity (Tappe et al. 2006). In another series of experiments, knockout of Homer1a exacerbated, and viral-mediated over-expression of Homer1a in the spinal cord dorsal horn attenuated, neuropathic pain hypersensitivity following chronic constriction injury (Obara et al. 2013). Consistent with these findings are the results of a pharmacological study wherein chronic intraspinal application of [−]-huperzine A, a potent and reversible acetylcholinesterase and NMDAR antagonist, prevented Homer1a up-regulation and retained GluA2-containing, calcium-impermeable AMPARs at synaptic sites following chronic constriction injury (Yu et al. 2013). On a behavioral level, [−]-huperzine A treatment resulted in a diminished pain phenotype (Yu et al. 2013). Thus, the NMDAR-mediated up-regulation of Homer1a in the spinal cord may represent an intrinsic mechanism by which

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neurons can curb their excitability and calcium responsiveness, and poses an interesting possible target for future pain therapies.

P2XRs P2X purinoreceptors are ligand-gated, ATP-sensitive, nonselective cation channels. Their activation leads to channel opening, and can result in the downstream stimulation of VDCCs and calcium-dependent MAP kinases (Cruz and Cruz 2007; Kaczmarek-Hajek et al. 2012). In particular, peripheral sensory neurons, but also neurons within the spinal cord dorsal horn, express P2XRs, including the P2X1, P2X2, P2X3, and P2X4 subtypes, and exhibit increased excitability in response to stimulation by ATP (Kim et al. 2003; Kuroda et al. 2012). Indeed, ATP not only functions as a pronociceptive stimulus by activating peripheral nociceptors but it is also released by sensory neurons at synapses in the spinal cord where its stimulatory action ultimately leads to an increased net spinal cord output (Jarvis 2010). A link between the development of peripheral and central sensitization in inflammatory and neuropathic pain models and the increased activity and expression of P2XRs is well established (DonnellyRoberts et al. 2008; Fabbretti 2013). Intracellular calcium rises are likely to be an important trigger for the regulation of P2XRs. P2X 3Rs, for instance, are sensitized by PKCdependent signaling pathways downstream from nerve growth factor (NGF) receptors (D’Arco et al. 2007; Giniatullin et al. 2008) and by the calcium/CaM-dependent serine protein kinase, CASK (Gnanasekaran et al. 2013). Moreover, brain-derived neurotrophic factor (BDNF) signaling through calcium, CaMKII, and cyclic AMP response element binding protein (CREB) has been observed to upregulate the expression of P2X3Rs in trigeminal sensory neurons (Simonetti et al. 2008). Interestingly, the co-activation by ATP of P2Y GPCRs, which can trigger the release of calcium from internal stores, may result in reduced P2XR responses due to a loss of plasma membrane phosphoinositides, suggesting that ATP acting on peripheral sensory neurons may simultaneously evoke algesic and analgesic responses (Bernier et al. 2013; Gerevich et al. 2007; Song and Varner 2009). The potential contributions of P2XRs to neuronal pain signaling are two-fold. On the one hand, P2XR activation, which can be triggered by concentrations of ATP down to the nanomolar range, results in neuronal depolarization and increased excitability, and provides a well-documented means by which ATP can activate neurons in pain pathways (Fabbretti 2013; Jarvis 2010). On the other hand, P2XR-derived calcium signals may contribute to the mechanisms involved in pain sensitization, for example by reducing GABA receptor-dependent inhibition (Shrivastava et al. 2011; Sokolova et al. 2001).

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VDCCs While it is generally accepted that VDCCs regulate fast neurotransmission and neuronal excitability in peripheral and central pain pathways, the specific channel subtypes involved, and the mechanisms by which this regulation occurs, vary across both cell types and pain modalities (Bourinet et al. 2014). LVA (T-type) VDCCs, for example, play a demonstrated role in peripheral as well as central and supraspinal pain processing and sensitization (Bourinet et al. 2005; Nelson and Todorovic 2006). T-type VDCCs are activated near the resting membrane potential and evoke low-threshold calciumdependent spikes or action potentials that can, in turn, trigger bursts of action potentials. Consequently, the activation of Ttype channels on neuronal dendrites is an efficient signal for the transduction of painful stimuli (Cheong and Shin 2013; Todorovic and Jevtovic-Todorovic 2011). Indeed, both the conduction of painful stimuli in peripheral sensory nerves and the gating of sensory information at the level of the thalamus have been reported to depend on T-type VDCC activity (Bourinet et al. 2005; Cheong et al. 2008; Cheong and Shin 2013; Todorovic and Jevtovic-Todorovic 2011). However, while T-type channels in the periphery seem to be involved in pronociceptive functions, in the thalamus they may exert anti-nociceptive effects (Todorovic and Jevtovic-Todorovic 2011). Moreover, although the expression of these channels following chronic constriction injury is upregulated in the spinal cord, their activity is nearly completely lost in sensory fibers (McCallum et al. 2003; Wen et al. 2006). Thus, while the intrathecal application of T-type VDCC antagonists or the down-regulation of their expression in the spinal cord using antisense oligonucleotides was observed to relieve both allodynia and hyperalgesia following chronic constriction injury (Wen et al. 2006), this pain model was associated in sensory neurons with a nearly 60 % reduction of pharmacologically isolated T-type currents and an 80 % reduction in total calcium influx (McCallum et al. 2003). These results support the idea that, in neuropathic pain, the up-regulation of T-type VDCCs is important in the mechanisms of spinal central sensitization, while their downregulation may be involved in peripheral sensitization (Hogan 2007; McCallum et al. 2003; Wen et al. 2010). HVA (L-, N-, and P/Q-type) VDCCs undergo two major changes in peripheral sensory neurons in chronic pain models. On the one hand, peripheral inflammation and spinal nerve ligation have both been observed to trigger a decrease in HVA calcium currents in these cells (Fuchs et al. 2007; Lu et al. 2010; McCallum et al. 2011; Rycroft et al. 2007). This loss of whole-cell calcium currents leads to the decreased stimulation of small (SK) and large (BK) conductance calcium-activated potassium channels, a reduction in action potential-evoked afterhyperpolarizations, and a consequent increase in neuronal excitability (Bahia et al. 2005; Lirk et al. 2008; McCallum

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et al. 2006; Sarantopoulos et al. 2007; Zhang et al. 2012). In particular, the loss of VDCC-mediated calcium currents is associated with a broadening of action potentials, an elevated maximal firing rate, and a higher incidence of burst firing (Amir and Devor 1997; Hogan 2007; McCallum et al. 2006). Both action potential duration and burst firing were demonstrated to be key determinants of neurotransmitter release and synaptic strength (Lisman 1997; Sabatini and Regehr 1997). Therefore, a decrease in VDCC-mediated calcium currents in nociceptive fibers represents a proximal cause for the increased neurotransmitter release onto and excitability of second order neurons within the spinal cord dorsal horn (Hogan 2007). Supporting this idea are observations that aberrant action potential waveforms and burst firing are directly triggered by antagonism of VDCCs or intracellular calcium buffering, and that the hyperexcitability of nociceptive afferents in chronic pain models can be corrected by a restoration of calcium influx (Hogan et al. 2008; Hogan 2007; Lirk et al. 2008). On the other hand, painful nerve injury is accompanied by an increase in the expression of the α2δ1 auxiliary subunit of HVA VDCCs (Dolphin 2013), and spinal nerve ligation and acute inflammation have both been associated with the increased expression of N-type VDCC protein in afferent sensory neurons and at synaptic locations in the spinal cord dorsal horn (Bourinet et al. 2014; Cizkova et al. 2002; Lu et al. 2010; Yokoyama et al. 2003). HVA VDCCs are multimeric protein complexes formed by the assembly of a pore-forming (α1) subunit together with two auxiliary (β and α2δ) subunits. The α2δ subunit is linked to the plasma membrane via a glycophosphatidylinositol anchor, and is complexed with the extracellular face of the pore-forming subunit (Bourinet et al. 2014). Importantly, α2δ subunits participate neither in pore formation nor in the gating of VDCCs. Rather, inclusion of this subunit in HVA VDCCs seems to have regulatory functions: that of a chaperone which aids in the trafficking of HVA VDCCs to synaptic membranes, and that of a potentiator in VDCC-exocytosis coupling (Davies et al. 2007; Hoppa et al. 2012). Consequently, the increased expression levels of α2δ1 observed in chronic pain models is consistent both with the documented increased trafficking of N-type VDCCs to presynaptic sites and with increased presynaptic neurotransmitter release probability and postsynaptic excitation (Bauer et al. 2009; Bourinet et al. 2014; Hoppa et al. 2012; Li et al. 2014a; Lu et al. 2010). In fact, the antinociceptive action of the drug gabapentin, which binds to α2δ subunits, is thought to derive from its ability to disrupt VDCC trafficking to presynaptic sites (Davies et al. 2007; Hendrich et al. 2008). Consistent with the idea that the up-regulation of α2δ1 may be a proximal cause for pain sensitization is the observation that the exogenous over-expression of α2δ1 results in enhanced VDCC activity in sensory neurons and hyperexcitability in their targets within the spinal cord dorsal horn (Li et al. 2006).

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Moreover, α2δ1 knockout animals exhibit significantly delayed development of mechanical hypersensitivity in a nerve injury model of neuropathic pain (Patel et al. 2013). Further, blockade of injury-induced spinal nerve activity diminished increases of α2δ1 expression in the DRG, and abolished its up-regulation at synaptic sites in the spinal cord dorsal horn. These changes were correlated with a delayed onset of allodynia following injury (Boroujerdi et al. 2008). Along the same lines, knockdown α2δ1 expression by antisense oligonucleotides reverses allodynia in spinal cord injury and spinal nerve ligation models of neuropathic pain, and in a chronic constriction injury model in the trigeminal nerve (Boroujerdi et al. 2008, 2011; Li et al. 2004, 2014a).

RTKs and GPCRs One major source of calcium in neurons, besides the channels that allow calcium entry from the extracellular space, is the ER. Internal calcium release from the ER into the cytosol is triggered by the activation of phospholipase C (PLC)-linked RTKs and Gq/11-interacting GPCRs that are coupled via the activation of PLC to the production of diacylglycerol (DAG), which together with calcium stimulates protein kinase C (PKC), and inositol trisphosphate (IP3), which triggers the subsequent IP3R-dependent intracellular release of calcium (Berridge 1998; Rhee 2001). Plasma membrane receptors that can evoke the release of calcium in neurons along pain pathways include the NGF and BDNF RTKs, TrkA and TrkB, respectively, P2Y purinoreceptors, group I/II mGluRs, serotonergic (5-HT) receptors, noradrenergic receptors, opioid receptors, and many others (McKelvey et al. 2013; Stone and Molliver 2009; Trang et al. 2011). RTKs and GPCRs, and the calcium rises and calcium-dependent signaling pathways they activate, stimulate a broad range of ion channels in peripheral and central pain pathways, including calcium-permeable acidsensing ion channels, TRP channels, NMDARs, and P2XRs, but also calcium-sensitive chloride and potassium channels (Jin et al. 2013; Kweon and Suh 2013; McKelvey et al. 2013; Schicker et al. 2010; Stone and Molliver 2009). Indeed, the molecular and cellular consequences of RTK and GPCR activation and internal calcium release are established mediators of peripheral and central sensitization, and as such represent attractive candidates for potential pain therapies (Liu and Salter 2010; McKelvey et al. 2013). P2Y receptor activity on peripheral sensory neurons, for example, can sensitize TRPV1 channels and inhibit currents through P2XRs and N-type VDCCs (Borvendeg et al. 2003; Gerevich et al. 2005; Moriyama et al. 2003), and is linked to increases in the phosphorylation of MAP kinases and to elevated neuronal excitability deriving from a decrease in potassium channel expression (Li et al. 2014b). Moreover, the activity of P2YRs and the calcium rises they evoke have been shown to

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make an important contribution to the development of neuropathic and inflammatory pain sensitization (Li et al. 2014b; Malin and Molliver 2010). Pain-modulating descending pathways represent a particularly interesting source of GPCR-stimulating neurotramitters. There exist both inhibiting and facilitating descending pathways, but the serotonergic/noradrenergic pathway and the opioidergic pathway represent the best-characterized sources of descending pain modulation. The dual modality of these descending pathways is thought to be due to the existence of two distinct populations of neurons in the brainstem, “on cells” and “off cells,” which are differentially recruited by higher brain structures in conditions of chronic pain, stress, or fear to facilitate or inhibit pain at the spinal level (Heinricher et al. 2009). Adding to this complexity is the fact that both inhibitory (Gi/o-coupled) and excitatory (Gq/11coupled) GPCRs for descending modulatory neurotransmitters are expressed in the spinal cord dorsal horn (Millan 2002). Thus, the consequences of neuromodulator release will clearly depend on the receptor subtype that is activated. It follows that the relative expression levels of the different receptor classes and the types of neurotransmitter released by each fiber modality may determine the consequences of a given neuromodulatory input. Moreover, nociception-associated changes in relative receptor expression levels thus represent an attractive mechanism for changed responses to descending modulatory inputs. GABAergic interneurons of the spinal cord dorsal horn, for instance, were recently shown to express a much greater proportion of Gq/11-coupled (5-HT1A) than of Gi/o-coupled (5-HT2A) serotonin receptors (Wang et al. 2009). Thus, even during chronic pain states, when the expression of these two serotonin receptor types is increased (Zhang et al. 2001, 2002), serotonergic input onto these cells is likely to lead to their increased activity, and consequently to a net decrease in spinal excitation (Wang et al. 2009).

RyRs RyRs, ER calcium release channels of which the endogenous agonist is calcium itself, can amplify calcium rises originating either intracellularly or at the plasma membrane. RyRs are likely to play a role in the transduction of pain signals by amplifying presynaptic calcium rises leading to neurotransmitter release (Huang et al. 2008; Ouyang et al. 2005). Recently, RyR activity was shown to participate in pain chronicity by stimulating the calcium-/CaM-dependent protein kinase CaMKII in nociceptive neurons (Ferrari et al. 2013). The RyR-dependent amplification of calcium signals both pre- and postsynaptically has also been proposed as an indispensible trigger for the induction LTP at C-fiber synapses in the spinal cord dorsal horn (Cheng et al. 2010; Lu et al. 2012; Naka et al. 2013). RyR-mediated internal calcium

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release is implicated, as well, in pain processing along the thalamocortical pain pathway (Cheong et al. 2011), and in the mechanisms involved in central analgesia (Galeotti et al. 2005, 2008). Importantly, SERCA function and ER calcium content are reduced in sensory neurons following spinal nerve ligation and in animal models of diabetic neuropathy (Duncan et al. 2013; Gemes et al. 2009; Kruglikov et al. 2004; Rigaud et al. 2009; Zherebitskaya et al. 2012). While these findings are consistent with a role for dysregulated internal calcium release in pain sensitization, they also point toward calcium dysregulation-mediated ER stress as a proximal mechanism for neurodegeneration and the development of pain sensitivity in disorders involving peripheral nerve damage (Austin 2009; Fernyhough and Calcutt 2010).

PMCAs PMCAs represent the major high-affinity calcium extrusion mechanism in neurons, and have been identified as one of the primary regulators of presynaptic calcium at sensory neuron synapses in the spinal cord (Shutov et al. 2013). PMCA activity in primary sensory neurons is subject to regulation by rises in intracellular calcium, prolonged action potential trains, and TRPV activation, all of which accelerate PMCAmediated calcium efflux (Pottorf and Thayer 2002). The mechanisms underlying PMCA up-regulation, which might serve to limit the amplitude and duration of calcium rises in hyperactive neurons, are controlled by calcium, and mediated by CaM and PKC (Werth et al. 1996). In light of the consequences of heightened synaptic activation for the transmission of painful stimuli and for transitions to sensitized pain states, it is not inconsequential that up-regulated PMCA activity in sensory neurons promotes hyperexcitability, possibly by limiting the calcium-dependent activation of potassium and chloride channels (Gemes et al. 2012). Interestingly, PMCA expression in second order neurons within the spinal cord dorsal horn may be down-regulated in chronic pain states (Tachibana et al. 2004), and loss of PMCA expression in spinal cord neurons is linked to neuronal damage and cell loss (Kurnellas et al. 2005).

NCXs NCXs are expressed on the plasma membrane of neurons throughout the central nervous system and in cell bodies and free nerve endings of sensory neurons (Canitano et al. 2002; Persson et al. 2010). Interestingly, NCXs have two possible modes of operation depending on the electrochemical gradient of the substrate ions. Under basal conditions, they catalyze the uphill transport calcium ions out of the cell and import sodium

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ions down their concentration gradient (forward mode). In contrast, when the plasma membrane is depolarized or when intracellular sodium concentrations are high, NCX operates in reverse mode, mediating the export of sodium and the import of calcium (Blaustein and Lederer 1999; Brini and Carafoli 2011). This feature makes the NCX a potentially very interesting subject for study in the context of the increased neuronal excitability associated with pain sensitization in general, and the sustained depolarization associated with nerve damage in particular. More specifically, although NCX typically operates in forward mode, the reverse mode of NCX operation in sensory neurons has been associated with neuropathic pain resulting from peripheral nerve injury (Kuroda et al. 2013; Muthuraman et al. 2008). Interestingly, NCXs have been localized not only at the plasma membrane of neurons but also in the inner nuclear envelope, where they may play a role in regulating nuclear and ER-associated calcium signaling (Ledeen and Wu 2007; Wu et al. 2009). The degree to which NCX and nuclear NCX contribute to pain sensitivity in neurons along pain pathways, and whether their expression and activity are altered in chronic pain states, remains to be investigated.

Mitochondrial MCU and NCLX The uptake and release of calcium ions by mitochondria, which is controlled by the activity of the MCU and NCLX proteins, respectively, represents an interesting mechanism for regulating the amplitude and duration of neuronal calcium rises and their downstream consequences (Drago et al. 2011; Shishkin et al. 2002). Indeed, calcium uptake and release by mitochondria has been proposed to control the duration of neurotransmitter release from sensory fibers in the spinal cord, where it has been identified as one of the primary regulators of presynaptic calcium transients, and to play an essential role in spinal LTP (Dedov and Roufogalis 2000; Kim et al. 2011; Medvedeva et al. 2008; Shutov et al. 2013). More specifically, the mitochondrial calcium store in capsaicin-sensitive DRG neurons was found to be functionally coupled specifically to calcium entry via TRPVs, and to facilitate the generation of long-lasting elevations in cytosolic calcium (Dedov and Roufogalis 2000; Medvedeva et al. 2008). These mitochondrially controlled, prolonged calcium rises were found, in turn, to enhance neuronal firing and neurotransmitter release (Medvedeva et al. 2008). Mitochondria were also found to buffer calcium rises resulting from DRG neuronal firing, and—together with PMCAs—to comprise one of the major presynaptic calcium clearance mechanisms in DRG/ spinal cord synapses (Shutov et al. 2013). Together, these and other findings suggest that mitochondrial calcium uptake and release control nociceptive neurotransmission in the periphery by influencing the excitability and activity of at least a

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subset of primary sensory neurons. Mitochondria are also implicated, however, in central nociceptive sensitization. In particular, the uptake by mitochondria of calcium entering the cell via NMDARs and their subsequent generation of reactive oxygen species was found to play a key role in the signaling pathways involved in spinal LTP, inflammatory hyperalgesia, and the altered GABAergic neurotransmission associated with neuropathic pain (Kim et al. 2011; Yowtak et al. 2011). Consistent with these observations, altered mitochondrial calcium homeostasis has been implicated in the pathogenesis of chronic pain, and is linked in particular to pain sensitivity in diabetic neuropathy (Fernyhough and Calcutt 2010; Hernandez-Beltran et al. 2013; Verkhratsky and Fernyhough 2008).

CaBPs Calbindin D28k is a cytosolic EF-hand-containing CaBP that is expressed throughout the central nervous system. Although historically considered to serve as a marker for inhibitory neurons (DeFelipe 1997), calbindin D28k is also expressed by glutamatergic neurons, and this expression can be altered by excessive neuronal activity (Carter et al. 2008; Magloczky et al. 1997). Calbindin D28k is expressed at all levels of the pain axis, including the amygdala, thalamus, and spinal cord dorsal horn, and in primary sensory neurons (Craig et al. 2002; Fournet et al. 1986; Goncalves et al. 2008; Ichikawa and Sugimoto 2002; Li et al. 2005; Rausell et al. 1992). Calbindin D28k immunoreactivity has also been observed in a small population of enkephalin-positive neurons in the trigeminal subnucleus caudalis, a brainstem nucleus associated with nociception at the level of the head and thought to be the functional equivalent of the spinal cord dorsal horn (Huang et al. 2011). Consistent with a role for calbindin D28k in nociception, knockout animals were shown to exhibit deficient GABAergic neurotransmission in this nucleus and reduced nociceptive responsiveness following cutaneous injection of formalin, a substance that caused inflammatory sensitivity and increased neuronal firing in control animals (Egea et al. 2012). Moreover, increased numbers of calbindin D28kpositive nerve fibers have been linked to inflammatory sensitivity (Barcena de Arellano et al. 2013), and the presence of a substantial number of newborn calbindin D28k-positive neurons have been observed in the amygdala in an animal model of neuropathic pain (Goncalves et al. 2008; Neugebauer et al. 2004). Calbindin D28k is of course not the only cytosolic CaBP likely to play a role in nociceptive plasticity. Two of the most commonly expressed CaBPs, parvalbumin and calretinin, but also secratagogin and neuronal calcium binding proteins 1 and 2, are currently under investigation as potential players in the mechanisms underlying chronic pain (Barcena de Arellano et al. 2013; Cao et al. 2011; Hughes et al. 2012;

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Pecze et al. 2013; Shi et al. 2012; Zacharova and Palecek 2009; Zhang et al. 2014).

Consequences of calcium signals Intracellular calcium rises in neurons regulate a broad spectrum of cellular processes, membrane proteins, and signaling cascades. Changes in the intracellular concentration of calcium ions can influence neuronal properties by modulating ion channel activity or localization (for instance via Homer1; see above); by regulating neurotransmitter release (for instance via the altered expression and activity of VDCCs; see above); by altering the activity of calcium-dependent enzymes, kinases, and phosphatases (Fields et al. 2005; Ji et al. 2007); and by inducing or down-regulating the expression of a wide variety of gene targets (Bading 2013; Flavell and Greenberg 2008; West et al. 2001). In light of the inhomogeneous subcellular distribution not only of calcium sources and sinks but also of calcium effectors, it is perhaps unsurprising that the physiological impact of a given type of calcium signal, or of its modification in a pathological pain state, clearly depends upon where in the cell this calcium signal occurs and from which source it derives. For instance, while a global increase in calcium influx through VDCCs in primary sensory neurons can limit neuronal excitability and thereby neurotransmitter release, a selective increase in calcium influx specifically through presynaptically localized VDCCs is likely to have the opposite effect. Thus, although primary sensory neurons express greater numbers of VDCCs in neuropathic pain models, the targeting of these channels through the α2δ subunit to presynaptic sites results in an increase, rather than a decrease, in postsynaptic excitation (Dolphin 2013; see above). Moreover, although it is by now well accepted that the development of pathological pain phenotypes requires modifications to gene expression, the subcellular localization and source of the instigating calcium signals involved determine which signaling cascades and transcriptional activators and repressors become activated, and thereby the specific pattern of genes that are affected (Bading 2013; Hagenston and Bading 2011). One particularly interesting type of neuronal calcium rise in this context is that which occurs within the nucleus (Bading 2013; Simonetti et al. 2013).

Nuclear calcium signaling Calcium rises in the nucleus can influence the transcription of specific transcription factor targets, for instance via the stimulation of calcium/CaM-dependent kinase IV (CaMKIV) and the activation of CREB and CREB binding protein (CBP), or via the activation and release of the transcriptional repressor downstream regulatory element antagonist modulator

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(DREAM) from transcription factor binding sites. Nuclear calcium can also regulate gene transcription on a global level via the modulation of DNA methylation or histone acetylation, for example by altering the activity of DNA methyltransferases or by promoting the nuclear export of histone deacetylases (HDACs) (Bading 2013; Hagenston and Bading 2011; Oliveira et al. 2012; Schlumm et al. 2013; Fig. 2). Many of the targets of nuclear calcium signals are implicated in central sensitization. The transcription factor CREB, for instance, is well accepted as an important player in long-lasting spinal cord plasticity (Kuner 2010). DREAM, originally identified as a negative regulator of the Pdyn gene, which encodes the analgesic opioid polypeptide prodynorphin, has been proposed as a mediator of gene transcription and spinal cord plasticity (Cheng and Penninger 2004). Indeed, DREAM knockout animals exhibit elevated dynorphin levels and markedly reduced responses in multiple pain models (Cheng et al. 2002). Some more recent reports identifying a long-lasting up-regulation of DREAM outside the nuclear compartment of spinal cord neurons in inflammatory pain suggest, however, that the modulatory influences of DREAM on pain may be additionally attributable to mechanisms apart from the regulation of gene transcription (Long et al. 2011; Zhang et al. 2007). A particularly intriguing function for calcium signaling in general, and for nuclear calcium signaling in particular, is the epigenetic regulation of gene transcription by histone acetylation and DNA methylation (Denk and McMahon 2012). Whereas histone acetylation leads to chromatin decondensation, the deacetylation of histones by HDACs results in chromatin condensation and diminishes transcription factor accessibility. Class IIa HDACs were recently identified as targets of nuclear calcium rises. In particular, antagonism of nuclear calcium signaling in primary hippocampal neurons was shown to inhibit the nucleocytoplasmic shuttling specifically of class IIa (but not class I or class IV) HDACs. Consistent with their known involvement in transcriptional regulation, over-expression or shRNA-mediated knockdown of class IIa HDACs was also found to alter the expression of a number of known nuclear calcium regulated genes, including two that encode proteins with a proven role in central sensitization, C1qc and Cox2 (Schlumm et al. 2013; Simonetti et al. 2013). Interestingly, HDACs—including members of class II —are up-regulated in the spinal cord following the induction of inflammatory pain and down-regulated in a model of neuropathic pain (Bai et al. 2010; Tochiki et al. 2012). Moreover, their pharmacological inhibition was shown to interfere with the induction and maintenance of thermal hyperalgesia and to restore GABAergic synaptic function in the spinal cord (Bai et al. 2010; Zhang et al. 2011). Importantly, our recent paper provides the first direct evidence that nuclear calcium signals in excitatory spinal cord dorsal horn neurons play an integral role in the mechanisms

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underlying central sensitization (Simonetti et al. 2013). More specifically, we observed that high-threshold, nociceptive-like stimulation (Torsney and MacDermott 2006) of sensory nerve fibers triggers nuclear calcium rises in spinal cord dorsal horn neurons (Fig. 2). Further, we found that the amplitude and duration of evoked nuclear calcium transients depends both on the frequency of the synaptic stimulation and on the putative number of recruited afferent axons (Simonetti et al. 2013). Thus, the increased firing rates and neurotransmitter release probability characteristic of nociceptive afferents in pathological pain states may be ideally suited to trigger nuclear calcium-dependent transcriptional responses involved in pain plasticity (Fields et al. 2005). Consistent with this idea is our finding that antagonism of nuclear calcium signaling using an exogenously expressed buffer for nuclear calcium/CaM, CaMBP4, effectively abrogates the activation of CREB, interferes with the up- or down-regulated expression of a wide range of pain-associated target genes, and disrupts the development of mechanical and thermal hypersensitivity in an animal model of chronic inflammatory pain (Simonetti et al. 2013). As outlined above, nuclear calcium signaling in spinal cord neurons is likely to regulate gene transcription and pain plasticity via multiple mechanisms (Cheng and Penninger 2002; Flavell and Greenberg 2008). Indeed, we also observed that the antagonism of CaMKIV via the overexpression of a kinase-dead CaMKIV mutant—unlike the antagonism of nuclear calcium signaling as a whole —disturbed the development of mechanical hypersensitivity, but not of thermal hypersensitivity, indicating that this pathway is differentially engaged in discrete pain modalities. Moreover, we found that only ∼30 % of genes whose expression is influenced by nuclear calcium signals in inflammatory pain are putative CREB targets (Simonetti et al. 2013). One of the primary candidate sources for nuclear calcium rises is the L-type VDCC (Hagenston and Bading 2011). Using calcium indicators expressed in the nucleus and replay of synaptic activity-evoked action potential trains, Bengtson and colleagues recently showed that L-type VDCCs in hippocampal neurons mediate at least 70 % of the amplitude of nuclear calcium rises evoked by bursts of synaptic activity (Bengtson et al. 2013). Although there is relatively little evidence implicating L-type VDCCs in the chronic pain plasticity, at least two recent studies have demonstrated that the expression of L-type VDCCs is altered in a spinal nerve ligation model of neuropathic pain (Favereaux et al. 2011; Fossat et al. 2010). In particular, these studies show that all three L-type VDCC subunits are up-regulated in chronic pain states, and that both the prevention of this up-regulation and the specific knockdown of L-type channels relieves pain hypersensitivity. They additionally observed that increased L-type VDCC expression on its own can trigger pain hypersensitivity (Favereaux et al. 2011; Fossat et al. 2010).

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Fig. 2 Nuclear calcium signaling in pain. a The nociceptive-like electrical stimulation of C-fiber afferents evokes nuclear calcium rises in excitatory neurons of the spinal cord dorsal horn. Left schematic of the spinal cord showing the imaged area and a wide-field fluorescence image of an acute spinal cord slice expressing the FRET-based nuclear calcium indicator TnXXL.NLS. Right fluorescence traces showing nuclear calcium rises triggered by suction electrode stimulation (100 Hz, 1 s, 0.8–2.0 mA) of the attached dorsal root (adapted with permission from Simonetti et al. 2013). Bar 50 μm. b Calcium entering the nucleus of neurons can regulate gene transcription by modifying the activity of a range of target proteins and transcription factors (TFs). Consequences of nuclear calcium signaling include transcriptional disinhibition via the transcriptional

repressor DREAM, the dissociation of methyl CpG binding proteins like MeCP2 from methylated DNA, the nucleocytoplasmic export of class IIa HDACs, and the activation of CaMKIV, which in turn can phosphorylate and activate numerous targets including CREB and its cofactor CBP. Examples of genes that are up-regulated and down-regulated by nuclear calcium signaling in excitatory neurons of the spinal cord dorsal horn in inflammatory pain include CaMKIIα, Grip1, Cox2, Pdyn, and Cacnα2δ3; and Timp1, C1qc, Ctsk, C3, and Ccl3, respectively. The nuclear calcium-dependent down-regulation of C1qc disinhibits spine growth and promotes the expression of an inflammatory pain phenotype (Simonetti et al. 2013)

Importantly, using antisense and siRNA strategies, Fossat and colleagues also established a clear link between the stimulation of L-type VDCCs, the phosphorylation of CREB, and the

enhanced transcription of Cox2 that is observed following nerve ligation (Fossat et al. 2010). On the basis of these findings, it seems possible that L-type VDCC-mediated

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calcium rises could represent a necessary component of excitation–transcription coupling at the spinal level.

Structural plasticity A key feature of peripheral and central sensitization is an increase in the strength of excitatory synaptic transmission at central synapses, and one of the primary mechanisms underlying this increase is a change in the density and size of synaptic spines (Kuner 2010). Rises in the intracellular concentration of calcium ions can serve as a proximal signal for such structural plasticity (Penzes et al. 2008; Simonetti et al. 2013). Several key transducers of calcium signaling that mediate pain hypersensitivity, including AMPARs and NMDARs as well as CaMKII and a number of other calcium-dependent kinases, have been shown to mediate spine stabilization and turnover, at least in brain circuits (Saneyoshi et al. 2010). In previous studies, neuropathic pain resulting from spinal cord injury was associated with increased de novo formation and elaboration of dendritic spines in spinal laminae IV and V (Tan et al. 2008). More recently, it was shown that peripheral inflammation can also lead to dendritic spine remodeling, and that this process is both dependent on calcium signaling to the nucleus and mediated by the repression of the complement cascade molecule C1q, which is known to trigger synapse elimination in mature and developing neurons (Chu et al. 2010; Ma et al. 2013; Schafer and Stevens 2010; Simonetti et al. 2013; Fig. 2). It is well established that cytoskeletal rearrangement is necessary for rapid dendritic spine plasticity, and that cytoskeletal modifications are linked to the enhanced insertion of calcium-permeable AMPARs into synapses during LTP (Fortin et al. 2010). One hypothesis is that an interaction between the AMPAR GluA2 subunit and Rac1, a member of the Rho family of GTPases, may serve to coordinate both the functional and structural plastic changes that accompany long-lasting plasticity (Asrar and Jia 2013). Several works have identified the Rho/Rac family of GTPases as key molecules involved in synaptic structural plasticity (Martinez and Tejada-Simon 2011; Schwechter et al. 2013). Indeed, these GTPases transduce signals coming from extracellular stimuli such as glutamate to the actin cytoskeleton and induce the clustering of AMPARs during spinogenesis (Schwechter and Tolias 2013; Wiens et al. 2005). Importantly, activation of Rho/Rac GTPases requires strong synaptic input, NMDAR activation, and calcium influx, and results in increased synaptic strength and synaptic spine size (Schwechter and Tolias 2013). Further, activation of Rac1 following spinal cord injury mediates changes in spine morphology and density in the spinal dorsal horn, and leads to injury-induced hyperalgesia (Tan 2008). Moreover, as discussed above, GluA2 AMPAR subunits are internalized in animal models of persistent inflammatory and neuropathic

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pain (Chen et al. 2013; Larsson and Broman 2008; Park et al. 2009), and the absence of GluA2 prevents the synapse loss that is otherwise observed following the mGluR-dependent long-term depression of synaptic strength (Nosyreva and Huber 2005). Therefore, the combined calcium-dependent up-regulation of Rac1 and internalization of GluA2 are likely to be permissive for structural plasticity in the spinal cord dorsal horn.

Outlook While it is well accepted that the amplitude, kinetics, and distribution of a given calcium rise are important for determining what its consequences might be (Hagenston and Bading 2011; Hardingham and Bading 2010; Raymond and Redman 2006), surprisingly little is known about the characteristics of synaptic activity-triggered calcium signals in distinct subcellular compartments, their contributions to cellular and functional plasticity, and whether and how they are altered in acute and chronic pain states. Moreover, although research into the mechanisms of chronic pain sensitization has expanded in recent years and includes investigations not only of excitatory neurons but also of astrocytes and microglia (Bardoni et al. 2010; Beggs and Salter 2010; Gao and Ji 2010; McMahon and Malcangio 2009), exceptionally little is known about their activation patterns in response to painful stimuli or about the calcium signals involved in these processes. Further, although intracellular calcium rises have been shown to be important for intercellular communication in activated astrocytes and microglia (Bardoni et al. 2010; Beggs and Salter 2010; Gao and Ji 2010; McMahon and Malcangio 2009), whether calcium signaling mediates transitions of glial cells from resting to activated states has not yet been examined. Finally, the characteristics of calcium signals in distinct types of neurons, both excitatory and inhibitory, throughout the entire pain axis have not been widely studied. A number of recent improvements in the properties of genetically encoded calcium indicators, however, should make possible the direct examination and characterization of cell-type- and subcellular compartment-specific calcium signals in chronic pain states (Akerboom et al. 2013; Ohkura et al. 2012). When employed in concert with pharmacological tools to modify specific ion channels or pathways, and/or with molecular tools that enable the knockdown or over-expression of select target proteins (Bengtson et al. 2010; Qiu et al. 2013; Simonetti et al. 2013), the use of such indicators offers a powerful means to explore the characteristics and consequences of global and subcellularly localized neuronal and glial calcium signals in pathological pain states.

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