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Distinguishing the Mechanisms of DRD2 Isomers in Reward-Seeking
Lake Forest College
Lake Forest, Illinois 60045
Addiction and reward-seeking grow increasingly prevalent in society with the increased availability of rewards. Hence, determining the molecular and genetic basis of reward-seeking is imperative in reducing the incessant craving for reward. While the DRD2 gene is associated with reward-seeking behavior, very little is known about the molecular functions of its receptor product—including whether the two alternatively spliced receptors are autoreceptors or heteroceptors and how this is linked to reward seeking. While knocking out each form alone has led researchers to determine the role each one plays in behavior and neural activity, distinguishing between the autoreceptor and heteroceptor function of each has yet to be conducted and linked to behavior via lox-cre mouse models.
The Phenotype: A brief overview
What do opioids, cat videos, chocolate, sex, shopping, beer, gambling, video games, and Netflix all have in common? These stimuli or behaviors are rewarding in that they incite appetitive wanting, are typically liked during consumption, and teach us to seek out these vices again and again. All of us have been guilty of “binge-watching” a TV show, taking that second piece of cake, or a throwing away a few hours on YouTube. Yet, some individuals are more likely to engage in reward-seeking behaviors than others.
Although each vice is different, there are a few commonalities amongst all compulsive reward seekers. The person constantly thinks of the object, activity, or substance and will engage in the behavior even if is causing harm to themselves or people close to them, such as a person whose addiction to gambling has cost him his life-savings. This is accompanied by a loss of control, in which the person cannot prevent themselves from engaging in the rewarding behavior, such as the women who binge-eats knowing she will develop health problems if she continues (American Addiction Centers, 2017). The person may also engage in the addictive behavior rather than something else they once enjoyed, and their relationship with others may be damaged as they repeatedly engage in the detrimental behavior. Finally, withdrawal occurs when they cease the activity; this may include irritability, craving, restlessness or depression.
While the causes of addiction are a mystery, the interaction of genes (inherited units of instruction) and the environment play a role. You may have noticed that alcoholism runs in families, for example. Of particular interest is a gene called DRD2, also known as the dopamine receptor 2 gene (D2R). Active in the brain, this gene provides the cell with the instructions to create a receptor that specifically receive a molecule termed dopamine. These neurotransmitter molecules are released by dopamine neurons and are thought to play a role in motivation or learning for a reward. There are multiple types of dopamine receptors, and these receptors have alternative functions separate from reward. D2R receptors can be found in reward centers within the brain, in regions such as the nucleus accumbens and dorsal striatum. The DR2 plays a role in attention, motor control, and motivation or learning about a reward (Lewis, 2013), so when the amount of DR2s is too low or too high, mental health issues like OCD, ADHD, and schizophrenia arise. Remarkably, when populations of DR2 are too low in reward-related areas of the brain, organisms are more likely to display addictive behaviors (Lewis, 2013).
Neuronal receptors are imperative for communication amongst brain cells. Embedded in the membranous border of the cell, receptors sit on the outside of the cell into synapses, small gaps between neurons into which neurotransmitter molecules are released. There, D2R has one of two functions, depending upon where the receptor is located (Usiello, 2000). They can be found on presynaptic dopaminergic neurons, which release dopamine into the synapse. (They are called presynaptic because in the timeline of neurotransmitter communication they are active prior to the synapse. They can also be found on “post-synaptic” neuronal cells, found on the other side of the synapse and receive dopamine that is released from the presynaptic cell.
When the receptor is located on presynaptic neurons, the receptors interact with dopamine that is release by their own cell—this type of receptor is called an autoreceptor. The autoreceptors allow the presynaptic neuron to learn about how much dopamine it has released and if it must stop or release more dopamine (Ford, 2014). In this way, DR2 autoreceptors regulate the release of dopamine from their own cells, activating a signal in their own neuron that stops the creation and release of DA so it is not always being made and released.
D2R can also be found on postsynaptic neurons, which do not necessarily build and release dopamine molecules, but have other functions in the brain. These postsynaptic neurons learn about dopamine release into the synapse when this dopamine interacts with postsynaptice D2R. These D2R become activated and start a cascade of communication, ultimately telling the postsynaptic cell to quiet down or to become more active. These D2R receptors are called heteroceptors since they do not regulate activity of the molecule the receive, incite other responses in the neuron in which they are embedded Beaulieu, 2011).
The DRD2 gene also makes two forms of the receptor so one is longer than the other. It was previously believed that the short form was an autoreceptor and the long form was a heteroceptor. Yet, these forms may be found on both presynaptic and postsynaptic neurons. Interestingly, both have divergent functions and may provide different information to the cell as both autoreceptors and heteroceptors. While autoreceptors and heteroceptor have been shown to play different roles in reward-seeking behavior, it is not clear how the short and long forms influence the cell to influence addictive-like behavior.
Molecular Function and Mouse Model
Two Distinct Functional Isoforms
According to omim.org, the DRD2 gene is found on the q arm of chromosome 11 in humans, at locus 11q.23.1 and is interrupted by 6 introns. The DRD2 gene encodes two different G-protein-coupled receptors (GPCR), both of which consists of seven membrane spanning segments whereby the carboxyl (C) terminal is intracellular and the amino (N) terminal is extracellular. The third intracellular loop is larger than the rest and cooperates with the Gi -protein. which are both linked to the Gi protein,hich subsequently inhibits adenylc clyclase and calcium channels, adjusting levels of cAMP.
Within the DRD2 gene, an 87-base-pair exon between introns 4 and 5 is alternately spliced into two different D2R isoforms, D2L (long form) and D2S (short form) (Usiello et al., 2000). These are both expressed simultaneously, but in a ratio favoring the long isoform, comprised of an additional 29 amino acids within the third intracellular loop of the GPCR (Beaulieu et al., 2011). Notably, these 29 amino acids confer different affinities for the Gi-protein, so that the D2L and D2S are functionally distinct receptors. These alternate functions were supposedly revealed in Usiello et al. (2000), who generated a mouse model in which the D2L isoform was selectively deleted by replacing exon 6 of DRD2 with a Pgk-neomycin cassette. Administration of haloperidol revealed that postsynaptic effects were impaired. Haloperidol, a D2-like antagonist, produces catalepsy by binding to the post-synaptic D2R and causes DA release from the presynaptic dopaminergic neurons. While haloperidol administration induced both of these effects in WT mice, catalepsy was not seen in the null-D2L mice, showing that the D2L isoform acted at postsynaptic sites and D2S served as a presynaptic autoreceptor.
However, this discovery sparked discord in the scientific community, as it was further shown that D2L and D2S isoforms could be both autoreceptors and heterosceptors. It has also been shown that the D2S receptors have a higher affinity for DA and but are more effective in inhibiting adenylc cyclase (Ford, 2014). However, D-2 receptors are found both on the terminals of DA neurons and post-synaptically on non-DA and it has thus been challenging to differentiate the roles of autoreceptors from that of heteroceptors and functions of D2L from that of D2S.
Notably, the autoreceptors and hetereceptors are also located in different regions of the brain. Development of bacteria artificial chromosome (BAC) mice that express green fluorescent protein (GFP) have revealed that medium spiny neurons selectively contain D2R autoreceptors that project from the ventral tegmental area (VTA) to the nucleus accumbens, a region of the brain related to motivation and craving (Bealieu et al., 2011). Here, autoreceptors regulate activity by activating potassium conductance and the expression of tyrosine hydroxylase, as DA precursor (Ford, 2014). Activation of these autoreceptors decreased excitability and release of DA and behaviorally resulted in subsequent decrease in mice locomotor activity.
Bello et al., (2011) and Anzalone et al., (2012) have attempted to distinguish between these two populations by creating knock-out model in which D-2 receptors have only been deleted from DA neurons, while retaining normal levels of D2-receptors in other areas to elucidate the role solely in reward-seeking, separable from the other roles of the D2 receptor (Ford et al., 2014). Anzalone et al., (2012) utilized cre recombinase to selectively remove receptors from either the substantia nigra and ventral tegmental area or from medium spiny neurons (MSN) in the striatum. While the loss of presynaptic autoreceptors in MSNs did not affect motor activity, these mice displayed hyperactivity in response to novel environment and increased cocaine sensitivity. This was not seen in autoreceptor deletion in the nucleus accumbens. Thus, the D2R autoreceptors in the nucleus did accumbens did not play as great a role in regulating DA and reward-sensitivity. However, activation of postsynaptic receptors has also resulted in increased dopamine release and increase locomotion (Bealieu et al., 2011).
Neve et al., (2013) furthered these results by creating D2 null mice and restoring expression of either the D2L or D2 receptor via virus-mediated receptor restoration in either MSN or within the nucleus accumbens. The authors found that in null-mice, deletion of both receptors striatal MSNs prevented autoreceptor functions responses such as quinpirole-induced inhibition of locomotor activity, inhibition of firing rate of the substantia nigra neurons, and inhibition of dopamine release. When the D2L receptor was restored, these activities resumed but not when the expected D2S receptor was restored. This study suggested that D2L and D2S may both be capable of acting as postsynaptic receptors and autoreceptors, and prior assumption attributing these autoreceptor functions to the D2S receptor may be incorrect.
Notably, very little has been shown about how the distinct isomers actually interact with the G protein itself, or even the role of the D2 receptor in certain pathways. However, evidence suggests that autoreceptor activation moderates and potassium levels to inhibit presynaptic DA release (Hill et al., 2001.).
Role of D2- Receptors in Reward-Seeking Behavior
Behavioral researchers typically delete all D2R in reward-related areas of the brain to show that these receptors in general affect addictive-like behaviors. For instance, Kramer et al., (2011) overexpressed D2R in the nucleus accumbens and dorsal striatum, finding that stimulation of the nucleus accumbens resulted in slower clearance of the subsequently released DA and increased locomotion. Importantly, when given cocaine, these rodents did not display sensitivity to cocaine, possibly because the additional cocaine-induced release of DA did not have an effect on the already saturated DA levels.
In addition, mice trained to lever press for food reward while lacking striatal D2 receptors showed distinct phenotypes. Researchers observed responding during extinction, in which animals were no longer rewarded for lever pressing with food. Compared to the WT mice, D2 mice stopped lever pressing much more slowly. This behavior displayed in the D2R knockouts demonstrate D2R is required for strong reinforcement. Further specifying the reinforcing functions of D2R, Bello et al. (2011) selectively knocked out D2 autoreceptors in the striatum. These mice displayed low basal levels of DA but potentiated responses to cocaine, via increased locomotion, lever pressing, and more time spent in a chamber associated with receiving cocaine. This again implicates D2 autoreceptors in the striatum as mediators of reward seeking but does not distinguish between distinct roles the different isomers as autoreceptors.
Notably, while the previous behavioral studies (Bello et al., 2011; Kramer et al., 2011; Soto et al., 2011) demonstrated a role of the D2R autoreceptors in reinforcing reward-seeking and promoting addictive-like behaviors, they did not distinguish between the two isomers, which have distinct functions and likely moderate reward-seeking behavior (Usiello et al., 2000 This may be because there are no agonists that selectively bind to D2L or D2S (Ford, 2014). Thus, the goal of the proposed study would be to distinguish the functional role of the two isomers that act as autoreceptors in the nucleus accumbens, with the hope of determining how these isomers influence reward-seeking. Hence, null D2 autoreceptor mice that lack these autorceptors only in the striatum will be generated, the isomers will separately be restored in these mice virus-mediated receptor restoration. Importantly, both behavior and molecular function will be assessed to link the two. In live mice, responding for food pellets following extinction will be observed to determine reinforcing effects of these particular isomers, and it is expected that D2L isomer will inhibit responding, suggesting a role for D2R in reward-seeking. Additionally, intracellular recordings will be conducted to elucidate how each type of isomer influences concentrations of calcium and potassium levels in striatal neurons. As little research has been done to explore these mechanisms, it is unclear what effects each isomer will have on intracellular concentrations of DAergic neurons.
While heteroceptors also appear to play a role in reward-seeking (Bealieu et al., 2011), the role of the autoreceptor is more established and Neve et al. (2013) showed that autoreceptor function in MSNs may be mediated by both isomers. However, the role of these isomers as autorecptors in the striatum, a specifically reward-related region, have not been differentiated. While Bello et al. (2011) selectively knocked out D2 autoreceptors, they did not distinguish between isomers. As autoreceptors may allow one to distinguish the functional roles these isomers have been shown to have different molecular roles, this study seeks to distinguish their effects in reward-seeking. Thus, focusing on distinguishing the roles of short and long forms of the autoreceptor will clarify the role of each isomer in reward-seeking.
First, as in Neve et al. (2013) mice without D2 autoreceptors will generated via cre recombinase, but this will be linked to a promoter expressed exclusively in the striatum so that the autoreceptors are knocked out in that reward-related region alone. The addition of a virus will selectively restore function of each isomer in this area. Controls will include both D2-null autoreceptor mice and WT littermates that have D2 autoreceptors, allowing one to distinguish between have both D2 autoreceptors, no D2 autoreceptors, and one of each isomer.
Following this restoration, a select number of animals will be sacrificed to conduct intracellular recording from single striatal DA neurons. As Hill et al., (2001) showed that D2R mediate potassium release and thus inhibit the hyperpolarized neurons, this an increase in potassium levels would hence regulate further DA release. However, as changes in calcium levels are required to induce release of DA neurotransmitter containing vacuoles, detecting a change in these areas would result in DA release. Application of exogenous DA to the D2R receptors would reveal whether they play a role regulating DA release. In WT, one would expect to detect increased calcium levels, as functional autoreceptors should DA release, while no change or an increase in calcium levels would be detected in D2-receptor null mice. Observing these changes would allow one to link these functions with behavior and determine whether continued DA release or cessation of released was related to reward seeking behavior.
Behaviorally, half of the animals in each of the four groups would first learn to lever press in response to a cue. The learning curves will be observed to determine if loss of these particular isomers effect learning. Only animals that reach asymptote will progress to the next stage to ensure one is measuring the reinforcing effects of behavior, not learning. Then, to determine the effects of knock-out mice on motivation, animals would then be required lever press for pellets at a progressive ratio schedule, in which the number of lever presses required to obtain the reward increase. The point at which animals stop responding will distinguish motivation between groups. This is a conceptual replication of Bello et al. (2011) but will use a paradigm similar to (Soto et al., 2016). As show in these studies, mice without autoreceptors will not show motivation for the reward, but WT will respond more. Whether the two isomers moderate these effects will be observed behaviorally.
Though dopamine has proven itself to be involved in reward, the specifics of the mechanisms still must be distinguished. While the DRD2 gene is related to reward-seeking and addiction, among others, it’s molecular and behavioral influence must still be mediated. Perhaps not so unfortunately, humans will continue engaging in these pleasurable, rewarding behaviors like eating cheese and watching cat videos.
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