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There is growing evidence for the role of voltage-gated L-type Ca2+ channels in mediating aspects of the addictive properties of psychostimulants. L-type Ca2+ channels activate Ca2+ second-messenger pathways that regulate protein phosphorylation and thereby activation of target gene expression. Here the authors will review recent progress in our understanding of L-type Ca2+ channel–activated signal transduction pathways that contribute to molecular neuroadaptations evident following acute and chronic exposures to psychostimulants.
The psychostimulants amphetamine and cocaine are the most reinforcing drugs known, both in humans and in animal models of drug addiction. These drugs exert their addictive effects by increasing synaptic levels of the neurotransmitters dopamine and glutamate in the reward pathway of the brain (Berke and Hyman 2000; Hyman and Malenka 2001). It is now well established that the life-long behavioral effects resulting from psychostimulant exposure are a result of molecular adaptations that alter normal brain function (Berke and Hyman 2000; Nestler 2001). Many such adaptations have been shown to result from altered regulation of protein phosphorylation and gene expression mediated by dopamine and glutamate, which activate intracellular second-messenger signaling pathways that contribute to both the short- and long-term effects of these drugs. In recent years, the importance of Ca2+ neurotransmission via the glutamate receptor subtypes, NMDA and AMPA, in the reinforcing properties of psychostimulants has emerged (Wolf 1998; Carlezon and Nestler 2002; Wolf 2002). In addition to these channels, another route of Ca2+ entry in neurons is the voltage-gated L-type Ca2+ channel (LTCC), an important mediator of neuronal plasticity (Deisseroth and others 2003; Groth and others 2003). There is growing evidence that LTCCs play an important role in contributing to the behavioral and molecular changes induced by psychostimulants (Licata and Pierce 2003; Rajadhyaksha and others 2004). This review will outline our current understanding of the molecular pathways activated by LTCC following acute and recurrent psychostimulant administration, with the goal of highlighting the importance of LTCCs as a prime candidate for subserving some of the short-term changes and long-term molecular adaptations induced by amphetamine and cocaine. We will focus our review on the mesoaccumbens and meso-striatal pathways that project from the ventral tegmental area (VTA) to the nucleus accumbens (NAc) and dorsal striatum, respectively, primary pathways implicated in the rewarding and habit-forming aspects of addiction (Everitt and Wolf 2002; Gerdeman and others 2003).
LTCCs belong to the family of voltage-gated Ca2+ channels (Ertel and others 2000) that form multisubunit complexes on the neuronal membrane (Fig. 1A) and are activated by membrane depolarization (reviewed in Catterall 1988). They are composed of a Ca2+ pore-forming Cav or α 1 subunit that exhibits voltage sensitivity. In addition, the complex consists of auxiliary β, α2-δ, and γ subunits that regulate the functional properties of the Cav subunit (reviewed in Walker and De Waard 1998). The Cav subunit is structurally composed of four homologous domains (I–IV) that are linked via cytoplasmic loops (Fig. 1B). Each domain consists of six transmembrane helices with the fourth helix serving as the voltage sensor for the channel. The Cav subunit also contains the binding sites for the pharmacological agents (dihydropyridines, phenylalkylamines, and benzothiazepines) commonly used to block and activate the channel. There are two subtypes of LTCCs formed in the brain: they either contain the Cav1.2 (α1C) or Cav1.3 (α1D) subunits. These two subtypes are activated and blocked by the same pharmacological agents and share a high degree of sequence similarity (Ertel and others 2000) except over a small region that corresponds to the variable region between the second and third transmembrane domains (Fig. 1B). LTCCs have traditionally been classified as high-voltage activated Ca2+ channels; however, the availability of subtype-specific antibodies, complementary DNA clones, and generation of knockout and transgenic mice has revealed that Cav1.2 and Cav1.3 have different physiological characteristics (Platzer and others 2000; Koschak and others 2001; Xu and Lipscombe 2001; Lipscombe 2002), neuronal and tissue distributions (Hell and others 1993; Rajadhyaksha and others 2004), and proteins they associate with at neuronal synapses (Olson and others 2005; Zhang and others 2005).
Activation of LTCCs results in the influx of intracellular Ca2+ that binds the Ca2+-sensitive regulatory protein, calmodulin (CaM; Fig. 2A), which integrates Ca2+ signals entering at the membrane with downstream activation of Ca2+/CaM-activated kinase and phosphatase pathways (Xia and Storm 2005). The two major kinase pathways activated by LTCCs are the Ca2+/CaM kinase and the mitogen-activated protein (MAP) kinase (ERK1/2) pathways (Deisseroth and others 2003). Both these pathways activate the transcription factor CREB and CREB-induced gene expression, critical mediators of psychostimulant-induced neuronal and behavioral plasticity (Berke and Hyman 2000; Nestler 2001). In addition, LTCCs also activate the Ca2+/CaM-dependent cal-cineurin (PP2B) pathway (Fig. 2A), a phosphatase pathway also important for neuronal and experience-dependent plasticity (Groth and others 2003).
Both Cav1.2- and Cav1.3-containing channels activate Ca2+ signaling pathway, but the functional specificity of the intracellular signal transduction pathways they activate is conferred by their existence as multiprotein signaling complexes (Fig. 2B and C). In general, these complexes have been found to consist of an anchoring or scaffolding protein that secures a kinase and a phosphatase close to the channel, allowing regulation of channel activity (Wong and Scott 2004). In addition, channels associate with downstream transduction molecules that link the channel to specific signaling pathways (Craven and Bredt 1998; Sheng and Pak 2000). Cav1.2 and Cav1.3 have been found to associate with different intracellular targeting molecules, suggesting differential contribution to neuronal signaling. Cav1.2 associates with the microtubule-associated protein MAP2B, a member of the A-kinase anchoring protein (AKAP) family of proteins (Davare and others 1999). Cav1.2 also associates with the catalytic subunit of the cyclic-AMPdependent protein kinase A (PKAc) and the phosphatase, protein phosphatase 2A (PP2A) (Davare and others 1999; Davare and others 2000), allowing regulation of Cav1.2 function via activity-dependent phosphorylation and dephosphorylation of a PKA site on the C-terminus of this subunit. In addition, Cav1.2 contains the PDZ interaction sequence, Val-Ser-Asn-Leu (VSNL), at its C-terminus that allows interaction of Cav1.2 with PDZ domain–containing proteins. Interactions via PDZ sites link channels to second-messenger pathways (Craven and Bredt 1998; Sheng and Pak 2000). To date, two PDZ domain proteins, channel interacting PDZ domain protein and neuronal interleukin-16, that interact with Cav1.2 have been identified in the brain (Kurschner and others 1998; Kurschner and Yuzaki 1999). This PDZ interaction site has been found to be critical for Cav1.2 activation of CREB protein phosphorylation and CREB-mediated gene expression (Weick and others 2003). Cav1.3 contains a PKA-dependent phosphorylation site, but the AKAP and associated kinase and phosphatases are not known (Fig. 2C). Cav1.3 also contains a PDZ interaction sequence, Ile-Thr-Thr-Leu (ITTL), albeit different from that of Cav1.2, which enables interactions with the synaptic scaffolding protein Shank (SH3 domain and ankyrin repeat containing protein). Shank in turn binds to the adaptor protein, Homer, which is involved in regulation of intracellular Ca2+ stores and found to regulate cocaine-induced behaviors (Szumlinski and others 2004).
In the sections below, we will focus on the role of LTCCs in molecular pathways activated by amphetamine and cocaine in the VTA, NAc, and dorsal striatum.
The VTA serves as the primary neuroanatomical site at which the molecular mechanisms that underlie long-lasting psychostimulant-induced addictive behaviors are initiated (Bonci and others 2003; Vezina 2004). The primary cell type in the VTA is the dopamine neuron, with its cell body and dendrites present in the midbrain and its axons projecting primarily to the NAc, dorsal striatum, and medial prefrontal cortex in the forebrain. Amphetamine and cocaine increase somatodendritic dopamine release in the VTA. Glutamate is released via activation of presynaptic D1 receptors and activates Ca2+ intracellular signaling pathways in VTA cell bodies that play a pivotal role in psychostimulant-induced molecular changes (Licata and Pierce 2003). In this section, we will cover the contribution of LTCCs in psychostimulant-activated Ca2+ second-messenger pathways in the VTA and discuss it in the context of its role in psychostimulantinduced behavior.
As shown schematically in Figure 3A, acute administration of amphetamine activates the Ca2+ second-messenger pathway in the VTA and has been found to increase CaM mRNA and protein (Michelhaugh and Gnegy 2000). Work from our laboratory has found that amphetamine activates the Ca2+-mediated MAP kinase (ERK1/2) pathway in VTA dopamine neurons (Rajadhyaksha and others 2004). We have further found that LTCCs are not involved in this acute response (Rajadhyaksha and others 2004). This observation is consistent with the presence of very low levels of Cav1.2 mRNA and protein, an activator of Ca2+-mediated ERK1/2 phosphorylation, in VTA dopamine neurons (Rajadhyaksha and others 2004). Two alternative routes of ERK1/2 phosphorylation in the VTA in response to acute amphetamine are via NMDA receptors or the neurotrophic factor neurotrophin-3, both important mediators of psychostimulant-induced behaviors (Pierce and others 1999; Sweatt 2001; Thomas and Huganir 2004).
As shown schematically in Figure 3B, LTCCs have been found to be necessary to induce some of the persistent neuroadaptations evident in the VTA following chronic psychostimulant administration (Licata and Pierce 2003; Rajadhyaksha and others 2004). At a molecular level, work from our laboratory has found that following chronic amphetamine treatment, LTCCs down-regulate ERK1/2 phosphorylation in VTA dopamine neurons, a change that occurs in parallel with an increase in Cav1.2 mRNA and protein (Rajadhyaksha and others 2004). The increase in Cav1.2 gene expression may be regulated by glutamate signaling, as the Cav1.2 gene contains the upstream regulatory sites cyclic AMP and calcium response element (CRE) and Activator Protein 1 (AP1), which bind the transcription factor CREB and the Fos family of proteins, respectively (Liu and others 2000). These regulatory sites and their transcription factors are activated by psychostimulants and glutamate signaling and contribute to some of the long-lasting changes induced by these drugs (Berke and Hyman 2000; Nestler 2001).
We have also found that the decrease in ERK1/2 phosphorylation is accompanied by an increase in calcineurin (PP2B) and MAP kinase phosphatase-1 (MKP-1), regulators of ERK1/2 phosphorylation. As LTCCs have been found to activate PP2B (Graef and others 1999; Chang and Berg 2001; Snyder and others 2003) and play a role in phosphatase-mediated neuronal plasticity (Groth and others 2003), we hypothesize that following chronic psychostimulant exposure, Cav1.2-containing LTCCs in VTA dopamine neurons are involved in activating a phosphatase pathway. This adaptation in the VTA may either represent a homeostatic response to chronic psychostimulant exposure, as has been ascribed to the role of MKP-1 (Bhalla and others 2002), or represent psychostimulant-induced neuronal plasticity via activation of phosphatases as observed for other experiencedependent neuronal events (Groth and others 2003). Figure 3 represents a schematic of our hypotheses. We speculate that one of the pivotal adaptations that occur in VTA dopamine neurons during the transition from acute to chronic psychostimulant exposure involves activation of the Cav1.2 gene and its Ca2+-mediated phosphatase pathway.
Ca2+ influx via LTCCs has been found to play an important role in psychostimulant-induced behaviors (Licata and Pierce 2003). In the VTA, the immediate component of acute psychostimulant-activated Ca2+ signaling is predominantly mediated via NMDA receptors, whereas the subsequent contribution of recurrent psychostimulantactivated Ca2+ signaling is additionally mediated by LTCCs. Consistent with this formulation is the fact that NMDA blockers inhibit both acute and chronic psychostimulant-induced behaviors (Karler and others 1989; Vezina and Queen 2000), whereas LTCC blockers inhibit only chronic, but not acute, psychostimulant-induced behavioral changes (Karler and others 1991; Pierce and others 1998). This finding is consistent with our molecular data identifying a role for LTCCs in chronic, but not acute, amphetamine-mediated ERK1/2 phosphorylation in the VTA (Rajadhyaksha and others 2004). Such findings are also consistent with the observation that MAP kinase (ERK1/2) pathway inhibitors do not block acute cocaine-induced behaviors but inhibit chronic cocaine-induced behaviors (Pierce and others 1999). This suggests that the LTCC-activated MAP kinase pathway in VTA neurons may be selectively activated following recurrent but not acute psychostimulant exposure. In addition, the increase in Cav1.2 mRNA and protein observed in the VTA following chronic amphetamine is consistent with the fact that psychostimulant-induced neuroplasticity requires new protein synthesis in the VTA (Sorg and Ulibarri 1995). Based on the above findings, we propose that Cav1.2 and ERK1/2 pathways in the VTA contribute to psychostimulant-induced neuronal plasticity that underlies aspects of the long-term alterations in behaviors evident following recurrent drug exposure.
The nucleus accumbens (ventral striatum) and caudate/putamen (dorsal striatum) represent the primary neuroanatomical sites in the brain at which psychostimulant-induced molecular changes are consolidated. It is these persistent and life-long molecular adaptations that underlie aspects of addictive behaviors, including withdrawal and craving leading to relapse, which may be seen even after long periods of drug abstinence (Hyman and Malenka 2001). Like in the VTA, the primary neurotransmitter pathways that are activated by amphetamine and cocaine in the NAc and dorsal striatum are the dopamine and glutamate pathways. Also like in the VTA, LTCCs mediate components of signaling through both these pathways and contribute to psychostimulant- induced behavior. However, much less is known about the precise contribution of the pathways activated by LTCCs in the NAc and dorsal striatum that result in psychostimulant-induced molecular adaptations.
Amphetamine and cocaine release dopamine that activates dopamine D1-like and D2-like receptor subtype second-messenger pathways (Berke and Hyman 2000). Dopamine D1 receptors activate the cyclic AMP (cAMP)/PKA pathway that phosphorylates LTCCs at a PKA-specific site, thereby increasing channel activity (Fig. 4; Surmeier and others 1995). Dopamine D2 receptors have been found to activate PP2B via the phospholipase C pathway, decreasing LTCC activity via PP2Bmediated dephosphorylation (Fig. 4; Hernandez-Lopez and others 2000). In the dorsal striatum, the phosphorylation state of LTCC is also regulated by the CaM binding protein, regulator of calmodulin signaling (RCS) also known as cAMP-regulated phosphoprotein (ARPP-21) (Rakhilin and others 2004). RCS serves as an integrator of Ca2+ and cAMP signals. When RCS is phosphorylated by PKA, it becomes an inhibitor of PP2B, one of its target molecules. Acute cocaine and the amphetamine derivative, methamphetamine, have been found to increase phosphorylation of RCS (Caporaso and others 2000). Based on this finding and the regulation of LTCC phosphorylation by RCS, Figure 4 represents our hypothesis of the acute effects of psychostimulants on LTCC phosphorylation state. We speculate that amphetamine and cocaine will increase phosphorylation of RCS, which in turn will inhibit PP2B activity, resulting in an increase in LTCC phosphorylation. This would then activate the LTCC-mediated Ca2+ second-messenger pathway and contribute to psychostimulant-induced activation of molecular pathways.
The most extensively studied pathway in the dorsal striatum, and to some extent in the NAc, is the dopamine and adenosine 3´, 5´-monophosphate–regulated phosphoprotein (DARPP-32) pathway (Greengard 2001). DARPP- 32 is a phosphoprotein that integrates the cAMP second-messenger pathway activated by dopamine D1 receptors and the Ca2+ pathway activated by NMDA and LTCCs. DARPP-32 modulates multiple downstream targets involved in psychostimulant-induced gene expression, particularly via the transcription factor CREB. LTCCs have been found to influence both dopamine and NMDA neurotransmission (Liu and Graybiel 1996; Rajadhyaksha and others 1999) and, by acutely increasing the levels of intracellular Ca2+ as well as by activation of kinases, play a direct role in contributing to activation of some of the downstream targets of these signaling pathways (see Fig. 5A). Although the precise role of the Cav1.2 versus Cav1.3 subtypes in contributing to psychostimulant-mediated molecular pathways has remained elusive, both of these subtypes have been shown to phosphorylate CREB (Dolmetsch and others 2001; Zhang and others 2005). Regarding the regional specificity of the Cav1.2 versus Cav1.3 subtypes in contributing to psychostimulant-mediated molecular pathways, a recent study using transgenic mice that allows specific activation of the Cav1.3 subtype has revealed that in the dorsal striatum, Cav1.2 activity is required for activation of CREB-induced Fos expression, whereas in the NAc, Cav1.3 activity mediates CREB-induced Fos expression (Sinnegger-Brauns and others 2004). This suggests that the Cav1.2 and Cav1.3 pathways may selectively contribute to the actions of psychostimulants in the dorsal striatum and NAc, respectively.
A clue into the possible role of LTCCs in contributing to the molecular changes that are sustained following recurrent drug exposure, even after long drug-free periods, has come from physiological studies using the behavioral sensitization model of drug addiction (Wolf 2002). Such animal models of addiction share many features of synaptic plasticity evident in animal models of learning and memory, including the induction of longterm depression (LTD), a phenomenon shown in the hippocampus to be mediated by persistent alterations in glutamatergic signaling, which has additionally been associated with decreased levels of intracellular Ca2+ and with increased phosphatase activity (Winder and Sweatt 2001). Likewise, it has been found that NAc neurons previously exposed to recurrent cocaine exhibit LTD (Thomas and others 2001) and exhibit persistent alterations in glutamatergic neurotransmission, as well as decreased basal intracellular Ca2+ and increased phosphatase signaling (Zhang and others 2002). As LTCCs (Snyder and others 2003) and amphetamine (Wang and Uhl 1998) have been found to acutely increase PP2B activity, respectively, we speculate that the contribution of LTCCs to psychostimulant-induced long-term synaptic plasticity in the NAc may be mediated by persistent activation of the Ca2+/CaM-dependent phosphatase pathway (Fig. 5B). This hypothesis is further supported by the observation that DARPP-32, a downstream target of PP2B, has been found to be dephosphorylated at the PP2B-specific site (Thr 34) in samples taken from the dorsal striatum and NAc 10 days following recurrent cocaine administration (Scheggi and others 2004).
In summary, the findings presented above establish an important role for L-type Ca2+ channels and its Ca2+ activated pathways in mediating some of the addictive properties of psychostimulants. However, there are many unanswered questions regarding the contribution of Cav1.2 versus Cav1.3 to the acute and chronic effects of psychostimulants. Future research on molecular correlates of addiction requires consideration of L-type Ca2+ channel signal transduction pathways in the VTA in initiating and in the dorsal striatum and NAc in sustaining some of the critical neuroadaptations underlying the transition from the drug-naive to drug-addicted state.