Running head: DOPAMINE MODULATION NEOSTRIATUM 1 Neostriatal Dopamine Modulates Motivation: Incentive Salience Generation in the Neostriatum by

Motivational processes are constantly at work to focus us on relevant stimuli for survival. The mesolimbic dopamine system has been strongly linked to motivation and reward-directed behavior, especially in regions such as the nucleus accumbens and ventral tegmental area. However, the nigrostriatal (substantia nigra to neostriatum) pathway has also been implicated in reward and motivation. Incentive salience is the motivational value placed on cues paired with reward. This study aimed to test whether increased dopamine in the neostriatum could enhance incentive salience of a learned cue. Differing levels of a D1/D2 dopamine agonist cocktail were combined with an autoshaping paradigm as a measure of incentive salience using both dorsomedial and dorsolateral neostriatum cannula placements. Results indicate that dopamine stimulation may increase incentive salience in the direction of predictive cues in the dorsolateral neostriatum but increase incentive salience in a more general fashion in the dorsomedial neostriatum. DOPAMINE MODULATION NEOSTRIATUM 3 Neostriatal Dopamine Modulates Motivation: Incentive Salience Generation in the Neostriatum Motivational processes are essential for our daily lives, but they often go unnoticed. If something is desired or needed, there is a good chance that a motivated attempt will be made toward the goal, whatever that goal may be. Many neural mechanisms have been implicated in the production and execution of motivation, both consciously and unconsciously. Classically, the mesolimbic dopamine system, including the nucleus accumbens and ventral tegmental area, has been viewed as the neural center for reward (Berridge, 2004). However, recent evidence has suggested that other brain structures may play an equally important role in reward processing. Throughout the last half century, it has become increasingly apparent that dopamine is the neurotransmitter most important for reward (Berridge, 2007; Dayan & Balleine, 2002; Wise 2009). However, only recently has evidence pointed to a specific role for dopamine within the scope of reward. Namely, dopamine is responsible for the „wanting‟ portion of reward, rather than „liking‟ and, to a lesser extent, learning (Berridge, 2007; Berridge & Robinson, 1998). „Wanting‟ refers specifically to the incentive salience process of motivation. Incentive salience is an association between natural rewards and learned stimuli that are paired in a Pavlovian fashion. The reward (an unconditioned stimulus) is paired with some sort of cue (a conditioned stimulus), and eventually the same motivational behaviors normally directed at the reward are then aimed at the cue as well. In this sense, the cue becomes „wanted‟ as much as the natural reward, and is attractive enough to warrant approach and consummatory behaviors normally directed at the reward (Berridge, 2007). Incentive salience is an anticipatory process, with the reward-paired cues causing motivational responses. These responses are locked to the moment of cue presentation, which is the moment that „wanting‟ can be observed. DOPAMINE MODULATION NEOSTRIATUM 4 A major site of dopamine innervation is the neostriatum, often referred to as the dorsal striatum or caudate putamen. It is a major crossroads of connectivity between the mesolimbic dopamine system and the neocortex (Thorn, Atallah, Howe, & Graybiel, 2010; Voorn, Vanderschuren, Groenewegen, Robbins, & Pennartz, 2004). Along with cortical projections, inputs from the substantia nigra, ventral tegmental area, and nucleus accumbens all converge in the neostriatum (Haber & Knutson, 2010; Yin, Ostlund, & Balleine, 2008). The neostriatum is traditionally viewed as a site of stimulus-response (S-R) learning, which involves the learning of strict responses to stimuli that can be classified as habits or rigid responses. However, there is growing evidence that dopamine in the neostriatum plays a role in motivation as well. Dopamine in the neostriatum has been proven necessary for motivated behaviors by depletion studies. A strain of dopamine-deficient mice, which show a complete lack of motivation for natural rewards and must be given L-DOPA daily for survival, show a rescue in eating behavior, locomotion, and reward-learning with the addition of a dopamine-promoting virus into the center of the neostriatum. Motivated behaviors are not rescued when the virus restores dopamine only to the nucleus accumbens, which may indicate that the neostriatum is essential for motivational processing and output while the nucleus accumbens modulates more particular aspects of motivation (Palmiter, 2008). This demonstrates the importance of the neostriatum in the manifestation of motivated behaviors and suggests that it is sufficient for the integration and output of motivated behaviors carried out by the dopaminergic subcircuit. Other studies have also indicated that neostriatal dopamine signaling is central to motivational processes. Evidence exists in reward learning, where substantia nigra neurons fire with almost identical patterns to ventral tegmentum neurons in the presence of stimuli paired with rewards (Hollerman & Schultz, 1998). Extracellular dopamine levels increase in the DOPAMINE MODULATION NEOSTRIATUM 5 neostriatum during presentation of a drug-paired cue, suggesting that the expectation of reward is sufficient to cause neostriatal dopamine release and has a larger role than simple motor response production (Ito, Dalley, Robbins, & Everitt, 2002; Robbins & Everitt, 1992). There is evidence that dopamine in the neostriatum, while involved in the execution of motor activity, also has a role in drug-seeking behavior. The blockade of dopamine receptors in the dorsal striatum inhibits cocaine-seeking behavior, with rats showing a reduced response to cocaine-paired drug cues (Vanderschuren, Di Ciano, & Everitt, 2006). In humans, the neostriatum has been shown to act in a similar fashion. Neostriatal dopamine release is evoked by the presentation of drug cues to cocaine addicts and can accurately predict feelings of drug craving (Volkow et al., 2006). This suggests that dopamine in the neostriatum plays an important role in drug craving, and subsequently the „wanting‟ portion of reward as described by incentive salience. One way to model incentive salience attribution in the laboratory is the autoshaping phenomenon, which involves the attribution of motivated responses to a conditioned stimulus paired with an unconditioned stimulus. With autoshaping, the UCS reward is delivered regardless of the effort put forth to receive it. This procedure results in the attribution of a conditioned response to the reward cue, such as a lever presented with a cue light before a food reward is given. Some animals will show approach and interactions with the cue object, while others are more inclined to interact with the area of reward presentation, such as a food cup. The animals that interact with the cue are called sign-trackers, and those that interact with the reward cup are called goal-trackers. For both of these groups behavior is locked to the cue presentation, which means that behaviors focused on the prepotent stimulus will only be enhanced during the presence of the reward-predictive cue. Even though the two targets differ, both may express incentive salience. DOPAMINE MODULATION NEOSTRIATUM 6 A classic example of the autoshaping phenomenon is observed by Brown and Jenkins (1968). Pigeons were presented with a lighted food tray that appeared for a four-second interval either before or after another lighted cue was presented. The group that experienced the cue light before the food tray was presented (forward pairing) began to approach and peck the cue light (Brown & Jenkins, 1968). Other examples of this phenomenon exist, such as a raccoon that was trained to put coins in a piggy bank and then rewarded as reinforcement. Eventually, the coins themselves became a sort of reward for the raccoon, as he would not let go of them, even if reward was no longer given (Breland & Breland, 1961). These reward-paired cues elicit a dopamine response, as has been illustrated in a great number of studies (Berridge, 2007; Berridge & Robinson, 1998; Ito et al., 2002; Robbins & Everitt, 1992). When investigating the role of dopamine in the neostriatum, it is important to note the different responses that dopamine signaling is capable of producing. Five types of dopamine receptors (D1-D5) are known, and these are divided into two families of receptors. The D1 family contains the D1 and D5 receptors and the D2 family contains the D2, D3, and D4 receptors. The striatum contains mostly D1 and D2 receptors, and D5 receptors to a lesser extent (Humphries & Prescott, 2010). One known function of dopamine is the regulation of glutamate signaling in medium spiny neurons, which are the most prevalent neuronal population in the striatum (Surmeier, 2007). This glutamate signaling in D1 receptor-expressing neurons has been shown to be necessary for the formation of associations between cue and reward in incentive learning processes (Novak et al., 2010). Although dopamine signaling and its subsequent cascades are much more complicated than simple excitation and inhibition, in general D1 receptors increase glutamate signaling and responsiveness postsynaptically (neuronal excitability), while D2 receptors seem to decrease downstream glutamate signaling (neuronal DOPAMINE MODULATION NEOSTRIATUM 7 inhibition) in the striatopallidal system through NMDA and AMPA receptor regulation (Palmiter, 2008; Surmeier, 2007). Since dopamine responses are somewhat complicated, it may be most useful to look at the effects of general dopamine excitation in the neostriatum before looking at the roles of individual receptor subtypes. The division between medial and lateral neostriatum has been identified by several studies. This division is based on both neural connectivity and function. In habit learning, the medial and ventral neostriatum show different patterns of activation during the learning of paired associations, and these differences also manifest themselves in the motivational output that they produce (Thorn et al., 2010). The largest distinction in connectivity and function seems to be along the ventromedial to dorsolateral striatal gradient. This would suggest that the dorsomedial and dorsolateral neostriatum also show a division of function, as the gradient is not the simple dorsal-ventral arrangement that was previously thought (Voorn et al., 2004). Lesions of medial and lateral portions of the neostriatum produce different behavioral effects, which illustrate that a division of function accompanies this topographical division (Yin et al., 2008). Additionally, connections between the cortex and neostriatum respond differently in terms of synaptic plasticity over a medial to lateral gradient. This could indicate that anatomical differences underlie the differences in functional output generated by both the medial and lateral neostriatum (Smith, Musleh, Akopian, Buckwalter, & Walsh, 2001). Overall, evidence is beginning to point to more specific areas of the neostriatum in regards to connectivity and function, so it is necessary to break the neostriatum down into smaller pieces to determine whether this division can be better explained. While it is obvious that dopamine is important in reward and plays a role in the function of the neostriatum, there are still areas in which dopamine is poorly understood. The following DOPAMINE MODULATION NEOSTRIATUM 8 set of experiments will address the question of incentive salience generation in the neostriatum, particularly by dopaminergic activation. To address this question, an autoshaping paradigm will be employed to test sign-tracking and goal-tracking behaviors in the presence and absence of a general dopamine receptor agonist (as well as a mu-opioid receptor agonist, used to verify an effect already observed in this laboratory [DiFeliceantonio & Berridge, 2010]). In addition, fos immunofluorescence will quantitatively measure specific neuronal activation under vehicle and dopamine agonist conditions. Most importantly, all experiments will be examining both dorsolateral and dorsomedial neostriatal cannulae placements to observe whether a medial/lateral separation of function is present in the neostriatum in respect to incentive salience processing.