Running Head: COCAINE- & NATURAL REWARD-INDUCED PLASTICITY 1

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1 Running Head: COCAINE- & NATURAL REWARD-INDUCED PLASTICITY 1 The Rewiring of Reward: A Comparison of Cocaine- and Natural Reward-Induced Synaptic Plasticity

2 COCAINE- & NATURAL REWARD-INDUCED PLASTICITY 2 Abstract A recently developed theory holds that drugs of abuse and natural rewards (such as food, sugar, and sex) converge on the mesolimbic reward system of the brain (Kelley & Berridge, 2002). Synaptic plasticity the modification of the connection strength between neurons occurs in this system, notably in the ventral tegmental area (VTA) and nucleus accumbens (NAc). Differences between drug- and natural reward-induced synaptic plasticity might account for the persistent and robust behaviors comprising drug addiction, including the susceptibility to relapse. This paper reviews research comparing the synaptic and structural neuroadaptations evoked in the VTA and NAc by cocaine and natural rewards, with a particular focus on recent and pioneering papers. Key Words: Addiction; Drug; Natural Reward; Synaptic Plasticity; Mesolimbic Dopamine System

3 COCAINE- & NATURAL REWARD-INDUCED PLASTICITY 3 The Rewiring of Reward: A Comparison of Cocaine- and Natural Reward-Induced Synaptic Plasticity The history of human society is closely intertwined with a narrative of drug use and abuse. Cocaine, a psychostimulant derived from Erythroxylum coca, was domesticated by the indigenous people of the western Andes almost 7,000 years ago (Saah, 2005; Sullivan & Hagen, 2002). The recreational use of cocaine and other psychoactive drugs has persisted to this day, and the abuse of such drugs is now considered a significant challenge to public health. In the United States, an estimated 22.5 million Americans use illicit drugs, with 1.4 million of these individuals specifically taking cocaine (Substance Abuse and Mental Health Services Administration, 2012). During 2011, 670,000 Americans used cocaine for the first time, and a significant proportion of these individuals can be expected to develop clinical dependence (Falck, Wang, & Carlson, 2008). In the current edition of the Diagnostic and Statistical Manual of Mental Disorders (the DSM-IV-TR), cocaine addiction falls under the broader category of substance dependence, and is characterized by the compulsive and repetitive use of a drug, despite negative consequence (American Psychiatric Association, 2000). Given the significant and widespread consequences of cocaine addiction, the neural mechanisms underlying its cause and maintenance are of critical scientific and medical interest. This paper aims to compare the synaptic plasticity or adaptations in the strength of signaling between neurons induced by cocaine with those evoked by natural rewards. Drug Addiction: A Disease of Neuroplasticity A major challenge in the treatment of drug addiction is the high risk of relapse into drug use, even after extensive periods of abstinence and after all withdrawal symptoms have

4 COCAINE- & NATURAL REWARD-INDUCED PLASTICITY 4 disappeared (Hyman, 2005). Relapse is often precipitated by the presence of drug-related cues internal or external stimuli which were previously associated with drug use (Gipson et al., 2013), such as the sight of drug paraphernalia. A prominent theory holds that this persistent vulnerability to relapse is preserved by similarly long-lasting changes to neural circuitry in the brain (Van den Oever, Spijker, Smit, & De Vries, 2010). Indeed, addiction seems a classic example of experience-dependent plasticity, in which long-lasting changes to behavior and psychological function are caused by an earlier experience in life (Robinson & Kolb, 2004). In the case of addiction, this experience represents not a singular instance of drug use, but rather a series of drug exposures. The neural systems which mediate memory and learning are commonly implicated in this plasticity (Hyman, 2005), due in part to the similarities between cue-induced relapse and associative learning processes. Under this perspective, addiction represents a pathological form of learning. This theory characterizes addiction as a disease of neuroplasticity, specifically affecting the normal processes of learning (O Brien, 2009). Neuroplasticity can occur at any number of levels, from adjustments in gene expression to the reconfiguration of neural networks, each of which can contribute to behavioral change. Synaptic plasticity which entails changes to the synapses, or connections between neurons is a critical mechanism for the modification of neural circuitry and could be responsible for some of the cognitive and behavioral features of cocaine addiction. A Connection between Addictive Drugs and Natural Reward Interestingly, the words addict and addiction were not commonly associated with drug use until the second decade of the 20 th century (Adams, 1935); in the period between the late 16 th and early 19 th centuries, the idea of addiction actually applied equally to drugs and

5 COCAINE- & NATURAL REWARD-INDUCED PLASTICITY 5 other perceived vices, such as sweet foods (Peele, 1990). This interpretation focused primarily on the gratifying properties of the addictive substance. These historical concepts of addiction foreshadowed contemporary controversy over the relationship between drugs and other behaviorally rewarding substances. In contrast with the colloquial association of reward with happiness and gratification, a reward in the behavioral sense is any object or event which induces approach behavior, consumption, and learning of these behaviors (Schultz, 2009). So-called natural rewards comprise substances and experiences such as food, water, and sex the consumption of which is evolutionarily beneficial (Kelley & Berridge, 2002). For several decades, it has been recognized that responses to natural rewards and addictive drugs share common features (Wise, 1980), including hedonic pleasure responses, motivated wanting, and associative learning of environmental cues (Hyman, Malenka, & Nestler, 2006). However, the magnitude which the brain often assigns too great a value to drug rewards, causing them to be pursued at the exclusion of other natural rewards or other activities. For example, cocaine-addicted individuals report a disproportionate valuation of drugs relative to food and sex, especially when describing the experience of being under drug influence (Goldstein et al., 2010). In addition, there seems to be a major difference between the two classes of reward in terms of biological effect (Kauer & Malenka, 2007). Natural rewards encourage behaviors that maintain biological homeostasis or encourage reproduction, both of which are evolutionarily advantageous; by extension, responsiveness to natural rewards is evolutionarily beneficial. Drugs of abuse, on the other hand, do not provide homeostatic or reproductive advantages and in fact are often harmful to personal health and functioning 1. Thus, even if there 1 In fact, due to the toxicity of many drugs at high doses, their natural originators psychotropic plant substances may have evolved to discourage plant consumption by herbivores (Sullivan, Hagen, & Hammerstein, 2008).

6 COCAINE- & NATURAL REWARD-INDUCED PLASTICITY 6 are underlying similarities between drug and natural reward, the effect of addictive drugs are in several respects distinctive from those of natural rewards. The Mesolimbic Dopamine System Examination of neural systems might reveal the mechanisms which cause these different effects. Reward processing is now widely believed to depend on the mesolimbic dopamine system, which consists of the ventral tegmental area (VTA), the nucleus accumbens (NAc), the amygdala, and several prefrontal areas (Spanagel & Weiss, 1999). As the name implies, the neurotransmitter dopamine is prominently featured in the mesolimbic pathway and has cultivated its own reputation as a neural encoder of reward (Schultz, 2009). Similarly, the VTA and NAc are two critical components of the mesolimbic dopamine system. The VTA is a heterogeneous collection of neurons located in the midbrain (Gutierrez, Lobo, Zhang, & De Lecea, 2011). It contains many of the brain s dopaminergic neurons, as well as smaller populations of GABAergic and glutamatergic neurons (Xia et al., 2011). The VTA is innervated by many brain regions, including the prefrontal cortex and the amygdala; the majority of these are excitatory glutamatergic inputs (Geisler, Derst, Veh, & Zahm, 2007). In turn, its dopaminergic neurons project to the NAc as well as associated limbic structures. In comparison, the NAc is a relatively homogenous group of neurons, located in the ventral striatum. It can be divided into two structures, the nucleus accumbens core (NAccore) and the nucleus accumbens shell (NAcshell) (R. Ito & Hayen, 2011). The NAccore and NAcshell diverge significantly in terms of both anatomy and function; for example, while the output systems of the two substructures overlaps somewhat, each additionally exhibits a distinctive pattern of regional connectivity. The vast majority (90% to 95%) of neurons in the NAc are medium spiny neurons (MSNs), which release the inhibitory neurotransmitter GABA as a chief

7 COCAINE- & NATURAL REWARD-INDUCED PLASTICITY 7 output (Grueter, Rothwell, & Malenka, 2012). MSNs are typically categorized based on the dopamine receptors they express; MSNs expressing D1 receptors (D1Rs) chiefly project to the midbrain as part of the direct pathway, while D2R-expressing MSNs project to the pallidal nuclei (the indirect pathway ). MSNs integrate excitatory, glutamatergic inputs from the PFC, amygdala, hippocampus, and thalamus, as well as the dopaminergic inputs from the VTA (Smith-Roe & Kelley, 2000). Forms of Synaptic Plasticity Broadly speaking, synaptic plasticity consists of any alteration in the synaptic connections between neurons; through alterations to the structure and strength of neuronal contacts, synaptic plasticity can lead to the rewiring of neural circuits (Holtmaat & Svoboda, 2009). For decades, prominent scientists like Santiago Ramón y Cajal 2 and Donald Hebb 3 theorized on the modification of interneuron communication, but no supportive evidence was produced until 1973, when Timothy Bliss and Terje Lømo reported the strengthening of synaptic transmission in the rabbit hippocampus (Berlucchi & Buchtel, 2009; Bliss & Lomo, 1973). This phenomenon, initially called long-lasting potentiation, eventually came to be known as longterm potentiation or LTP. Its counterpart the weakening of synaptic connectivity was discovered in 1982 and was termed long-term depression or LTD (M. Ito & Kano, 1982). These two classes of synaptic modifications have been intensely studied ever since their discoveries, and are now known as two of the cornerstones of synaptic plasticity. 2 Cajal proposed mechanisms of neuronal plasticity in his delivery of the 1894 Croonian Lecture of the Royal Society of London (entitled La fine structure des centres nerveux) (Jones, 1994). 3 In 1949, Hebb described lasting cellular changes caused by persistent activation: When an axon of cell A is near enough to excite a cell B and repeatedly or persistently takes part in firing it, some growth process or metabolic change takes place in one or both cells such that A s efficiency, as one of the cells firing B, is increased (Hebb, 1949, p. 62). This idea would profoundly influence later conceptions of synaptic plasticity (Cooper, 2005).

8 COCAINE- & NATURAL REWARD-INDUCED PLASTICITY 8 According to current understanding, the molecular mechanisms underlying LTP and LTD hinge on two glutamate receptors: α-amino-3-hydroxy-5-methylisoxazole-4-propionate acid (AMPA) receptor and N-methyl-D-aspartate acid (NMDA) receptor, also known as AMPAR and NMDAR. The AMPA and NMDA receptors are ionotropic their function is to allow the passive flow of ions through the neuronal membrane (Asztély & Gustafsson, 1996). LTP is triggered during the coordination of activity between the presynaptic and postsynaptic neurons. The presynaptic neuron releases glutamate, the primary excitatory neurotransmitter of the brain, into the synapse. The glutamate binds to both AMPARs and NMDARs on the postsynaptic membrane. Even when the NMDARs are bound by glutamate, ions cannot immediately flow through; magnesium ions block the receptor channel and are only displaced by sufficient postsynaptic depolarization (Kauer & Malenka, 2007). Thus, LTP is only triggered during the co-occurrence of NMDAR activation with postsynaptic depolarization. This causes an influx of Ca 2+ through the NMDAR channel, which initiates complex intracellular signaling cascades. Eventually, this signaling cascade alters AMPAR trafficking in the postsynaptic neuron, which in turn increases the number of AMPARs localized on the plasma membrane with no effect on NMDARs. The net increase of glutamatergic receptors at the synapse sensitizes the neuron to excitatory stimulation, resulting in larger postsynaptic responses. This form of LTP (known as NMDAR-dependent LTP) is the type of synaptic potentiation observed in the VTA and NAc; overall, there are numerous forms and variations of LTP, but as a whole they follow the same general pattern (Malenka & Bear, 2004). In contrast, LTD is initiated when weak stimulation of NMDARs co-occurs with a small rise in Ca 2+ levels in the postsynaptic neuron (Kauer & Malenka, 2007) 4. This activates an intracellular signaling cascade distinct from that involved in 4 A recent study by Nabavi and colleagues (2013) challenges the traditional belief that a small influx of Ca 2+ is necessary for LTD. The researchers demonstrated that ligand binding to NMDARs is sufficient to induce LTD in the

9 COCAINE- & NATURAL REWARD-INDUCED PLASTICITY 9 LTP, resulting in the removal of AMPARs from the synapse (again with no effect on NDMARs). Due to the net decrease in glutamatergic receptors on the synaptic membrane, subsequent excitatory stimuli generate smaller postsynaptic potentials. LTP and LTD primarily manifest through alterations in the number and properties of AMPA and NMDA receptors (Gerrow & Triller, 2010; Grueter et al., 2012). As a result, synaptic potentiation is routinely measured by examining the relative contributions of postsynaptic AMPARs and NMDARS to the excitatory postsynaptic currents (EPSCs) following presynaptic release of neurotransmitter. More specifically, it is measured as the ratio of AMPA receptormediated to NMDA receptor-mediated EPSCs, or AMPAR/NMDAR ratio. Thus, electrophysiological recording (which can probe the activity of synaptic receptors) is indispensable in assaying LTP and LTD (Okabe, 2013). The computation of AMPAR/NMDAR ratio begins with the electrophysiological measurement of the total whole-cell EPSC (Jeun, Cho, Kim, Li, & Sung, 2009). The AMPAR-specific current is then isolated by assaying EPSC in the presence of an NMDAR antagonist. The NMDAR-specific current is deduced by subtraction, and finally the AMPAR/NMDAR ratio can be calculated from the AMPAR- and NMDARspecific currents. Because NMDAR-mediated current is unaffected by LTP, an increase in the AMPAR/NMDAR ratio likely represents a corresponding increase in the efficiency and/or number of synaptic AMPARs (Ungless, Whistler, Malenka, & Bonci, 2001) and supports the occurrence of LTP. Similarly, a decreased AMPAR/NMDAR ratio implies a reduction in AMPAR efficiency and/or number. While changes in the number and function of synaptic receptors comprise an undoubtedly important component of synaptic plasticity, local structural changes to the synapse may also affect interneuron connectivity. This class of synaptic plasticity is known as structural plasticity absence of ion flux through NMDARs or increased Ca 2+ levels.

10 COCAINE- & NATURAL REWARD-INDUCED PLASTICITY 10 and specifically regards the neuronal structures known as spines. Spines are protuberances of the neuronal membrane, composed of a head connected to the dendrite by way of a thinner neck (Dietz et al., 2012). Dendritic spines express glutamate receptors (i.e., NMDAR and AMPAR) on their surface and receive more than 90% of excitatory synapses (Yuste & Bonhoeffer, 2001)(Dietz et al., 2012). Structural plasticity encompasses alterations in the density and morphology of dendritic spines, which can be caused by the remodeling of pre-existing spines as well as the creation of new spines. Because nearly all spines have a synaptic contact, increases in spine density and size are interpreted as increases in synaptic connectivity (Robinson & Kolb, 2004). Dendritic spines were first discovered in 1888 by Cajal 5 through the use of a tissue staining technique, the Golgi method (García-López, García-Marín, & Freire, 2007). A century later, the Golgi method is still commonly used to visualize the morphology of dendrites, as well as the density of dendritic spines (Robinson & Kolb, 2004). Technological developments such as electron microscopy allow the high-resolution quantification of structural plasticity (Nimchinsky, Sabatini, & Svoboda, 2002). However, these tissue staining and electron microscopy both present a static view of dendritic structure. The advent of modern digital microscopy, on the other hand, allows for imaging live synapses in vivo, allowing for a deeper understanding of synapse dynamics (Okabe, 2013). While electrophysiological techniques and staining/microscopy suggest two different sets of synaptic changes alterations to membrane receptors and dendritic structure, respectively accumulating evidence suggests that the two classes of plasticity often coincide. Spine enlargement and increases in postsynaptic spine density can often be observed simultaneous to 5 In his 1888 paper Estructura de los centros nerviosos de las aves, Cajal examined the surface of Purkinje cells and observed structures he describes as espinas cortas, or short spines (Cajal, 1888; García-López et al., 2007).

11 COCAINE- & NATURAL REWARD-INDUCED PLASTICITY 11 LTP (Engert & Bonhoeffer, 1999; Kauer & Malenka, 2007). Thus, although it is still valuable to assess each of these forms of synaptic potentiation in isolation, it appears as though LTP/LTD and structural plasticity are closely connected. A Comparison of Natural Reward- and Cocaine-induced Synaptic Plasticity Investigating Plasticity: Features of Experimental Design Rodent models are often used to capture aspects of drug addiction; for example, relapse into drug use can be modeled through the reinstatement of drug-seeking behavior in rats or mice (Gipson et al., 2013; Marchant, Li, & Shaham, 2013). Similarly, studies of synaptic plasticity typically focus on rats and mice. The results of these experiments seem to be critically impacted by aspects of experimental design, including the mode of reward delivery. The two most common modes of reward delivery are experimenter administration and self-administration. Experimenter-administered rewards, whether drug or natural, reveal changes that are evoked unconditionally the results of direct biological action in the brain. This protocol is sometimes alternatively referred to as non-contingent, because the delivery of reward is not contingent on any actions of the animal. Self-administered rewards, on the other hand, induce operant conditioning of behavior. Because self-administration experiments incorporate this form of learning, some of the observed effects may reflect neural mechanisms of operant conditioning, rather than the direct effects of the reward substance (Robinson & Kolb, 2004). Of the two procedures, self-administration is more reflective of clinical addiction because it resembles the voluntary behavior of drug-taking. However, the employment of both self- and experimenter-administered protocols is still productive. If both experimenter- and selfadministered reward produce the same magnitude and mode of synaptic plasticity, it is likely that the changes do not result from conditioning of the actions which preceded the experience of

12 COCAINE- & NATURAL REWARD-INDUCED PLASTICITY 12 reward. On the other hand, if different neuroadaptations result from the two protocols, learning processes are likely responsible for some of the self-administration effects. For example, a neuronal subpopulation might potentiate as part of behavioral conditioning by the experience of reward. The involvement of Pavlonian conditioning the learning of associations between two stimuli, such as a reward and a neutral environmental cue is more difficult to assess because such learning can occur in both contingent and non-contingent procedures. Ventral Tegmental Area: Natural Reward- & Drug-induced Plasticity In 2001, a seminal paper by Ungless and colleagues demonstrated that cocaine could alter glutamatergic synaptic transmission in the VTA (Ungless et al., 2001). In an experimenteradministered study design, mice were injected with a single dose of cocaine; subsequently, the researchers measured the AMPAR/NMDAR current ratio for VTA dopamine neurons. They observed a significantly higher AMPAR/NMDAR ratio in cocaine-treated mice than in both saline-injected and untreated mice. This potentiation could be observed 5 days after the exposure, but disappeared by the 10 th day. Ungless and colleagues found that currents induced by the injection of AMPA were potentiated by the single cocaine exposure, while the drug treatment had no effect on NMDA-induced currents suggesting an increased presence and/or efficiency of synaptic AMPARs, with no corresponding change to NMDARs (consistent with the effects of LTP). Later experiments have shown that the selective removal of NMDARs selectively on dopaminergic cells in adult mice blocks this potentiation (Zweifel, Argilli, Bonci, & Palmiter, 2008), increasing the evidence that the synaptic strengthening under observation is NMDARdependent LTP. The cocaine treatment produced no difference in AMPAR/NMDAR ratios in hippocampal cells or in GABAergic neurons of the VTA, demonstrating that the cocaine-induced potentiation is a specific response of the dopaminergic VTA cells. Overall, this study showed

13 COCAINE- & NATURAL REWARD-INDUCED PLASTICITY 13 that a single, passive injection of cocaine transiently strengthens the excitatory synapses of dopaminergic VTA neurons in a manner consistent with that of LTP. Argilli and colleagues (2008) followed a similar single-exposure procedure in order to determine how early LTP induction occurs in the VTA following exposure. Their results confirmed an increased AMPAR/NMDAR ratio in cocaine-treated rats; furthermore, using a microinjection of cocaine into the VTA, they demonstrated that cocaine can potentiate AMPARmediated transmission through local action in the brain. LTP in the VTA was detectable as early as 3 hours after the introduction of cocaine into the brain, and persisted for a period of 5 but not 10 days. These two studies established the rapid yet transient progression of VTA synaptic plasticity following a single exposure to cocaine. A number of studies have assessed VTA activity, but not synaptic potentiation, following a single episode of natural reward consumption. For example, it has been demonstrated that food ingestion activates dopaminergic VTA neurons in rats (Park & Carr, 1998). However, no researchers have investigated whether the effects of a single natural-reward experience (e.g., brief consumption of sucrose or a single sexual experience) induces LTP in the VTA. However, the selective in vivo activation of dopaminergic VTA neurons produced the same AMPAR redistribution as non-contingent cocaine administration (Brown et al., 2010). Since natural rewards are known to stimulate dopaminergic VTA neurons 6 (K. M. Kim et al., 2012), it is possible that they induce this very same redistribution of AMPA receptors. In fact, Parkitna and Engblom (2012) argue that the LTP-like plasticity observed in the VTA occurs whenever dopaminergic VTA neurons are stimulated sufficiently for the release of dopamine. This interpretation presents an interesting possibility, but it should be considered with caution the 6 Palatable meal ingestion activates neurons in the rat VTA (Park & Carr, 1998); similarly, sexual behavior activates dopaminergic neurons in the VTA of male rats (Balfour, Yu, & Coolen, 2004).

14 COCAINE- & NATURAL REWARD-INDUCED PLASTICITY 14 activation of neurons and their potentiation are quite different phenomena. As described previously, synaptic strengthening necessitates a specific combination of pre- and postsynaptic events beyond the activation of the postsynaptic neuron. In summary, a series studies has demonstrated that a single exposure to cocaine triggers rapid-onset yet transient LTP in dopaminergic VTA neurons. There is some evidence to believe that LTP-like potentiation occurs following a single experience of natural reward, but this class of synaptic plasticity has not been adequately studied. After all, investigation of the similarities and differences between natural reward- and cocaine-induced synaptic plasticity demands a thorough understanding of the timing and robustness of such neuroadaptations. Under the hypothesis that the effects of chronic drug use might be stronger than those of a single exposure, Borgland and colleagues examined the synaptic consequences of repeated cocaine administration (Borgland, Malenka, & Bonci, 2004). This chronic treatment protocol involved the experimenter-administered injection of cocaine into rats over 7 consecutive days. The experimenters found that repeated cocaine injection produced the same magnitude of synaptic strengthening as a single cocaine exposure; both single- and repeated treatment resulted in increased AMPAR/NMDAR ratio due to an upregulation of AMPARs on the synaptic membrane. Much like the synaptic plasticity following a single administration of cocaine, the LTP triggered by repeated exposure was transient, returning to normal levels by the 10 th day after cocaine treatment. A later experiment by Chen and colleagues was the first to directly compare mechanisms of synaptic plasticity shared by drug and natural reward (Chen et al., 2008). While prior studies primarily relied upon experimenter-administered, non-contingent reward, their investigation included the self-administration of various rewards. Rats were trained to self-administer cocaine,

15 COCAINE- & NATURAL REWARD-INDUCED PLASTICITY 15 food, or sucrose for two weeks. One day after the last administration, electrophysiological recording was used to test for potentiation. Rats that learned to self-administer food and sucrose displayed an increased AMPAR/NMDAR ratio equal to that observed in rats which selfadministered cocaine. But while the potentiation following self-administration of food and sucrose was transient disappearing by the third week following the last reward administration the synaptic strengthening caused by cocaine self-administration was detectable after 90 days of drug abstinence. In addition, this persistent potentiation precluded future synaptic adaptations to other stimuli. Notably, the extensive duration of plasticity was only observed under the selfadministration protocol; when cocaine was delivered in a non-contingent fashion (irrespective of rat behavior), no potentiation was observed. These results suggest that cocaine-induced synaptic plasticity might be generated in part by operant conditioning. Thus, this experiment provided several major breakthroughs. First, learning to self-administer either cocaine or natural reward induces LTP in dopaminergic VTA neurons. However, while the natural reward-induced adaptation is transient, cocaine-induced plasticity is persistent lasting for at least 3 months and excludes potentiation in response to subsequent stimuli. Second, the pharmacological effects of cocaine do not account for the entirety of cocaine-induced synaptic plasticity in dopaminergic neurons; these changes are induced in part by learning (e.g., operant conditioning). While dopaminergic neurons are undoubtedly a central feature of the brain s reward circuitry, the other neuron subtypes in the VTA also play important roles in responses to reward. GABAergic neurons account for 35% of the VTA neuronal population and are known to inhibit local dopaminergic neurons (Tan et al., 2012). This inhibitory function can be strengthened by the LTP of GABAA receptor synapses on dopaminergic neurons (also known as LTPGABA)

16 COCAINE- & NATURAL REWARD-INDUCED PLASTICITY 16 (Nugent, Penick, & Kauer, 2007). It has been suggested that LTPGABA generally co-occurs with the LTP of excitatory synapses on dopaminergic neurons as a homeostatic mechanism to control the neuronal firing rate (Kauer & Malenka, 2007). However, investigation has also revealed that repeated experimenter-administrated exposure to cocaine suppresses local GABAergic function (Liu, Pu, & Poo, 2005). The magnitude of GABAA receptor-mediated inhibitory postsynaptic currents (IPSCs) on local dopaminergic neurons was reduced following the drug regimen. Thus, cocaine might lead to an overall increase in reward signaling by preventing LTPGABA. If LTPGABA normally occurs during natural reward consumption to prevent the excessive activation of the dopamine system, the suppression of LTPGABA by cocaine offers a possible mechanism for the difference in effects of drug and natural rewards. This compelling theory merits additional investigation. While there is a proliferating body of literature on synaptic potentiation in the VTA, relatively little attention has been given to structural plasticity in VTA neurons (Russo et al., 2010). A study by Sarti and colleagues (2007) offered the first examination of cocaine-induced changes to spine density in VTA neurons. A single injection of cocaine consistently increased the spine density (and AMPAR/NMDAR ratio) in a subpopulation of VTA neurons. Since the neurons which experienced a surge in spine density also exhibited an increase in AMPAR/NMDAR ratio, it is a strong possibility that LTP and structural adaptations coincide in dopaminergic VTA neurons. No studies have been undertaken on the ability of natural reward to change spine morphology or density, making it impossible to determine whether natural and drug rewards both induce structural plasticity in VTA neurons.

17 COCAINE- & NATURAL REWARD-INDUCED PLASTICITY 17 Nucleus Accumbens: Natural Reward- & Drug-induced Plasticity Studies on plasticity in the NAc have focused on MSNs, due to their regional prominence as well as their critical involvement in mesolimbic dopamine circuitry. MSNs integrate a wide range of cortical and limbic inputs and account for the output of the NAc (Witten et al., 2010). MSNs are quiescent cells, so their activity depends heavily on these excitatory inputs and dopaminergic innervation from the VTA. Extensive observation of cocaine-induced plasticity in the NAc has demonstrated that it progresses on a slower timescale than plasticity in the VTA. And unlike in the VTA, a single administration of cocaine is not sufficient to induce synaptic plasticity in the NAc a longer period of cocaine exposure is required to produce neuroadaptations in MSNs (Mameli et al., 2009). In addition, withdrawal from drug exposure appears critical to synaptic plasticity in the NAc. For example, while the experimenter-administered injection of cocaine for five consecutive days does not alter the AMPAR/NMDAR ratio, the exact same drug regimen does induce a change in AMPAR/NMDAR ratio if it is followed by a two-week withdrawal period (J. Kim, Park, Lee, Park, & Kim, 2011). On this basis, many studies of the NAc test for synaptic potentiation after extensive drug-free or withdrawal periods. Over the last two decades, a number of studies have demonstrated that repetitive cocaine exposure depresses excitatory synaptic connectivity in NAc MSNs, under both experimenterand self-administration designs (Kauer & Malenka, 2007; Wolf & Ferrario, 2010). However, evidence that consistent administration of cocaine could evoke LTP in NAc MSNs also accumulated over this period. This empirical contradiction was not resolved until an experiment by Kourrich and colleagues in The researchers hypothesized that this difference existed because studies supporting the occurrence of LTP assayed electrophysiology during a drug-free,

18 COCAINE- & NATURAL REWARD-INDUCED PLASTICITY 18 abstinent period, while those suggesting a long-term depressive response to cocaine administered an additional challenge dose of cocaine at the end of the drug-free period before taking measurements (Kourrich, Rothwell, Klug, & Thomas, 2007). To test this idea, they compared two protocols of chronic cocaine administration followed by drug abstinence in MSNs. The sole difference between the protocols was the delivery of a challenge injection of cocaine in one design but not in the other. Results supported the experimenters hypothesis: abstinence following repeated exposure to cocaine evokes significant potentiation of AMPAR-mediated synaptic signaling, but the injection of a challenge dose reduces AMPAR surface expression, causing a shift to synaptic depression. This reduction in AMPAR surface expression occurs rapidly, becoming detectable 24 hours after cocaine re-exposure (Ferrario et al., 2010). Thus, it appears as though AMPAR-mediated synaptic transmission is strengthened in periods of abstinence following chronic cocaine experience, but quickly becomes weakened if cocaine abstinence ends. As evinced in the observations of Kourrich and colleagues, AMPARs have been identified as the major locus of LTP in the NAc (Wolf, 2010). An increase in synaptic levels of AMPAR on MSNs occurs consistently and robustly during withdrawal from repeated cocaine exposure. In contrast, NMDAR synaptic expression does not appear to change during withdrawal (Ferrario et al., 2010). These changes are detected by electrophysiology as an increase in the ratio of AMPAR- to NMDAR-mediated currents at the synapse. One recent study supported the ability of chronic natural reward exposure to cause this same form of synaptic potentiation. In rats, repeated daily ingestion of sucrose over the course of a week potentiated NAccore synapses through increased trafficking and expression of postsynaptic AMPAR (Tukey et al., 2013). This potentiation was also induced by ingestion of a noncaloric sweetener (saccharin), suggesting that

19 COCAINE- & NATURAL REWARD-INDUCED PLASTICITY 19 the plasticity is induced by a sort of sweet reward rather than the substance s caloric content. Although the AMPAR-mediated LTP in sucrose-exposed rats resembles cocaine-induced potentiation of MSNs, there is a critical difference between the timing of sucrose and cocaine effects. The synaptic changes evoked by sucrose were detected immediately after the reward regimen was completed. In contrast, extensive withdrawal periods are necessary for the induction of cocaine-induced LTP; AMPAR-mediated potentiation does not occur immediately after repeated cocaine administration (J. Kim et al., 2011). This topic is further complicated by the finding that following 2 week-long periods of food restriction (Peng, Ziff, & Carr, 2011) and sexual abstinence (Pitchers et al., 2010), the number of synaptic AMPARs in the NAc increases, mirroring both the design and results of cocaine exposure/abstinence studies. These discrepant findings might represent differences between individual rewarding substances and their respective effects; in order to better understand these inter-reward differences, the valence and time course of each substance s effects should be further investigated. In addition to LTP and LTD, another class of synaptic plasticity has been observed in the NAc: the formation of silent synapses. Silent synapses are glutamatergic connections in which NMDAR are present and functional, while AMPARs are either absent or nonfunctional (Huang et al., 2009; Lee & Dong, 2011). Because currents mediated by AMPARs are therefore absent (only NMDAR-mediated responses can be consistently detected), these synapses are described as silent. Additionally, the un-silencing of these special synapses through the insertion of AMPARs into the synaptic membrane (and subsequent addition of stable AMPAR-mediated activity) can evoke LTP. While silent synapses are often found in immature neurons, they are uncommon in the brain once developmental processes have concluded. However, Huang and colleagues (2009) demonstrated that repeated cocaine exposure generates considerable levels of

20 COCAINE- & NATURAL REWARD-INDUCED PLASTICITY 20 silent synapses on NAcshell MSNs. Cocaine was administered by experimenters over a 5-day period. The researchers assayed NMDAR- and AMPAR-mediated activity in the MSNs and assessed patterns in the expression of NMDAR and AMPAR subunits. They found that chronic exposure to cocaine produces NMDAR-active/AMPAR-silent excitatory synapses in NAcshell MSNs, indicating the generation of silent synapses in the VTA. Pitchers and colleagues (2012) similarly tracked the expression of NMDAR and AMPAR subunits in rat MSNs during a period of abstinence from sexual experience. After mating for 5 consecutive days, rats underwent an abstinence period of 1 day, 1 week, or 1 month. The surface expression of NR1 (a subunit of NMDAR) increased at 1 day of abstinence, but subsequently decreased after 1 week. In contrast, the expression of GluA2 (a subunit of AMPAR) increased intracellularly after 1 day of abstinence and at the surface after 1 month. The AMPAR/NMDAR ratio diminished in a rapid and long-lasting manner; this change was detectable at all time-points. The experimenters suggest that the swift internalization of AMPAR subunits and accompanying reduction in potentiation reflect an initial formation of silent synapses. Following this interpretation, the disruption of repetitive sex reward rapidly evokes the generation of silent synapses in NAc MSNs paralleling the effects of chronic exposure to cocaine. Repeated cocaine administration has marked consequences for structural plasticity in the NAc. In general, repeated cocaine injection is followed by rapid and long-lasting increases in spine density, regardless of whether it is self- or experimenter-administrated (Dietz et al., 2012; Robinson, Gorny, Mitton, & Kolb, 2001; Robinson & Kolb, 2004; Russo et al., 2010). It does not seem to depend on the experience of withdrawal, although the elevated density is capable of persisting for several weeks in the absence of drug-taking (Robinson et al., 2001). Sexual experience exhibits parallel effects on spine morphology in MSNs, resulting in increased spine

21 COCAINE- & NATURAL REWARD-INDUCED PLASTICITY 21 density after a week of sexual abstinence (Pitchers et al., 2010, 2012). In contrast, food reward does not replicate this form of plasticity. Robinson and colleagues (2001) directly compared changes in spine density resulting from the self-administration of cocaine and food. Rats that self-administered cocaine exhibited increased spine density on MSNs; rats that learned to selfadminister food, on the other hand, did not exhibit structural plasticity (Robinson et al., 2001). There also seems to be a shifting pattern of spine morphology during cocaine exposure and withdrawal. An increase in the number of spines with thin morphology can be observed shortly following chronic cocaine administration (Russo et al., 2010). These thin spines are highly plastic, and it has been suggested that cocaine-induced silent synapses primarily form on them. After a period of abstinence passes, the number of mushroom spines surpasses that of thin spines (Shen et al., 2009), paralleling the return of synaptic AMPARs to detectable levels. Gipson and colleagues (2013) assessed both synaptic and structural plasticity in a direct experimental comparison of cocaine and sucrose self-administration. Rats learned to selfadminister either cocaine or sucrose by lever press over the period of 10 days; light and sound stimuli were temporally correlated with reward delivery so that they would be learned as rewardrelated cues. At the end of this period, the lever-press behavior no longer resulted in reward delivery or cue presentation, resulting in its eventual extinction. Later, the reward and rewardassociated cues were reintroduced; lever-press behavior was subsequently reinstated in the rats. The reinstatement of cocaine self-administration was accompanied by a rapid and transient increase in AMPAR/NMDAR ratio and spine size in MSNs. While sucrose self-administration was also reinstated, synaptic potentiation did not occur in the NAc. Like previous studies of cocaine challenge following abstinence, the results of this study suggest that drug relapse entails a set of neuroadaptations that are not shared with the reintroduction of natural reward-taking.

22 COCAINE- & NATURAL REWARD-INDUCED PLASTICITY 22 Reflections on Research: Limitations and Conclusions An entire body of empirical evidence shows that exposure to either cocaine or natural reward produces neuroadaptations in the VTA and the NAc, two critical components of the mesolimbic dopamine pathway. Although a single exposure to cocaine does not seem sufficient to produce any changes in the NAc, it is capable of transiently eliciting LTP in the dopaminergic neurons of the VTA. Longer regimens of cocaine and natural reward administration induce similarly brief periods of potentiation in the VTA, although experimental evidence suggests that the learning to self-administer reward can extend the duration of this LTP for cocaine, but not for natural reward. In the NAc, synaptic plasticity follows more complicated patterns depending on both past and present experience of reward. Synaptic transmission is often enhanced in MSNs during the abstinent period following reward consumption, but can also be depressed by reexposure. However, several of these patterns differ significantly between drug and natural reward. For example, a regimen sucrose self-administration can immediately potentiate synaptic transmission in the NAc, without the need for a period of withdrawal. In addition, cocaine selfadministration leads to long-lasting potentiation in the VTA which excludes the induction of other neuroadaptations, while natural reward self-administration evokes only transient LTP perhaps suggesting a possible mechanism by which individuals with drug addiction fail to respond to non-drug rewards and incentives. One particularly pressing limitation on these experiments is the general heterogeneity of neurons, even within the same brain region or subpopulation; the possibility of differential properties between anatomical subregion and neuronal subtype thus deserves additional attention. In general, it is well established that neurons of different classes and even neurons of the same type, but located in different brain regions can exhibit distinct cellular properties.

23 COCAINE- & NATURAL REWARD-INDUCED PLASTICITY 23 Studies on the NAc have specifically indicated that differences in the synaptic effects of repeated in vivo cocaine treatment between NAccore and NAcshell MSNs (Kourrich & Thomas, 2009), as well as between D1R- and D2R-expressing MSNs (J. Kim et al., 2011). Similarly, Sarti and colleagues (2007) observed two subpopulations of dopaminergic VTA neurons, only one of which underwent plasticity as a result of cocaine exposure. Future research should recognize that differential properties might affect plasticity, especially within the population of NAc MSNs. Overall, there is abundant room for additional research on cocaine- and natural rewardrelated plasticity. The induction of synaptic plasticity in the VTA and NAc by cocaine has been much more thoroughly studied than natural reward-related adaptations, and only recently has an interest in the direct comparison of drug and natural reward effects emerged. The structural plasticity evoked by natural reward has been particularly neglected, and further investigation of Nonetheless, the increase in studies direct comparing drug- and natural reward-plasticity is an incredibly positive trend, as in the last 5 years several studies have produced incredibly intriguing data (e.g., Chen et al., 2008; Tukey et al., 2013), notably the identification of key differences between cocaine- and natural reward-induced synaptic plasticity. These differences in plasticity reflect possible ways in which drugs evade the regulatory mechanisms which govern natural reward. Knowledge of these evasive strategies, in turn, would help explain the incredible divergence between human consumption of natural reward which is beneficial for the organism and drug-taking, which leads to pathological behaviors and negative outcomes. The continuing investigation of reward and synaptic plasticity is well-situated to cultivate a deeper understanding of drug addiction, its neural underpinnings, and possible methods for intervention.

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