RUNNING HEAD: DELTA FOS-B MECHANISMS IN NUCLEUS ACCUMBENS AFTER SEXUAL EXPERIENCE

Molecular Mechanisms Underlying Sexual Experience- Dependent Neuroplasticity of the Nucleus Accumbens after in Syrian Hamsters

Written by: David Sidibe Haverford College

Thesis Advisor: Laura Been Second Reader: Jennifer Lilgendahl Haverford College DELTA FOS-B MECHANISMS IN NUCLEUS ACCUMBENS AFTER SEXUAL EXPERIENCE Sidibe 2

Abstract: This study is an aims to clarify the role of delta fos-b in the reinforcement of sexual behavior in Syrian (Golden) hamsters. We used a one-way ANOVA to compare levels of delta fos-b expression from experienced animals to naïve animals, and males to females. Western blot analysis was used to quantify the amount of protein in both the nucleus accumbens (NAc) and the caudate (CP), a control brain area. We found a significantly greater amount of delta fos-b expression in the NAc in sexually experienced hamsters (both males and females) compared to naïve. In addition, there was no difference between sexually experienced males and females in overall delta fos-b expression in the NAc. Implications and future directions point towards further unraveling the unknown mechanisms of reinforcement of sexual behavior in hamsters through humans.

DELTA FOS-B MECHANISMS IN NUCLEUS ACCUMBENS AFTER SEXUAL EXPERIENCE Sidibe 3

Introduction:

Male and Female Hamster Sexual Behavior

Male and female sexual behavior in hamsters is comparable to sexual behavior in other rodents, but includes some unique features. The estrous cycle of the female hamster typically lasts 4 days, and includes ovulation, or the releasing of the egg as it occurs with humans and other mammals. During the ovarian cycle, hormones play a large role in facilitating mating behavior. Estrogen is released 48 hours prior to the beginning of ovulation, and is responsible for organizing the behavior that increases the females proceptivity to the male. Progesterone injections closer to the period of ovulation are thought to facilitate the sexually receptive behaviors (Siegel, 1985).

The sexual activity of the female can be split up into stages. The first stage is called the proceptivity stage, in which the female actively advertises to the potential mate that she is ready to mate (Siegel, 1985). Proceptive behavior is directed towards the male, and is designed to make him more likely to initiate sexual behavior with the female. According to Johnston (1979), typical female hamster proceptive behaviors include the release of pheromones, specifically the release of a scent from the vagina called “vaginal marking”. The next stage of female sexual behavior is the receptivity phase. This is the stage in which the female physically facilitates the male’s sexual behaviors. A typical behavior displayed during receptivity by the female is lordosis, a stereotyped posture in which the female arches her back and lifts her tail in order to facilitate the male’s mounting, and make her vaginal region more accessible (Siegel, 1985). DELTA FOS-B MECHANISMS IN NUCLEUS ACCUMBENS AFTER SEXUAL EXPERIENCE Sidibe 4

Male sexual behavior in rodents is characterized by the behaviors of mounting, intromission, and ejaculation. Before mounting, the male shows his interest in the female by sniffing and licking the vaginal region. He will then wait for the receptive behaviors of the female before mounting her. After mounting the female, the male usually performs a series of thrusts in an attempt to get an intromission. Intromissions are insertions of the penis into the vagina without ejaculation of semen (Siegel, 1985). The male must perform several intromissions before an ejaculation is reached. The male then dismounts and repeats this process multiple times. According to Hull & Dominguez (2007), male hamster copulatory behavior is much quicker than rat copulatory behavior. It is characterized by an intromission every 10 seconds, and a post ejaculatory resting period that ranges from 35 seconds to 90 seconds (Siegel,

1985). In between each ejaculatory session (during the post ejaculatory interval), the male hamster will prepare for its next mount through licking of its genital region. The hamsters typically have 9-10 ejaculations before they are completely satiated and end their mating session with the female.

There are specific behaviors practiced during sexual activity in hamsters that make them an ideal animal to study when trying to find neural changes in the brain as a result of sexual experience. One characteristic of the Syrian hamster is that the female hamster has maintains lordosis throughout the mating session (Siegel, 1985), whereas other female rodents do not maintain lordosis, and are mobile during mating. This has methodological benefits, as less movement within the female can reduce confounds when trying to associate sexual behavior with specific molecular changes. In addition to moving little during the sexual experience, female hamsters play an active role in sexual activities. In the female hamster, there is a region near the DELTA FOS-B MECHANISMS IN NUCLEUS ACCUMBENS AFTER SEXUAL EXPERIENCE Sidibe 5

anus and the vagina called the perineal body. By anesthetizing the perineal body, males showed a

decreased success of intromission and a lower number of ejaculations, leading to an overall decreased level of successful mating (Noble, 1980). In addition, naive males paired with sexually experienced females had a higher percentage of successful intromissions after mounting the female compared to naïve males paired with naïve females, presumably due to the female’s sexual experience (Bradley, Haas, & Meisel, 2005). With a more active role in sexual experience, we would expect that the structures involved in sexual behavior will be activated more frequently.

In addition to these advantages, both male and female Syrian hamsters have been used as a model organism for many different studies (Meisel, Sterner, & Diekman, 1988; Meisel, Camp,

& Robinson, 1993; Bradley & Meisel, 2001; Schulz et al, 2004; Bell, Meerts, & Sisk, 2010).

Since there are many experiments that involve sexual experience that utilize Syrian hamsters as subjects, there exists a large literature and theoretical framework in which to place our results.

The advantage of this is that we can substantiate previously existing evidence, and contribute more to a field that is growing in research.

Mesolimbic Pathway in Sexual Experience –

The mesolimbic pathway, also known as the reward pathway, is an important neural

pathway involved in rewarding behaviors in hamsters and other mammals. Activation of the

mesolimbic pathway occurs through the activation of dopaminergic neurons in the ventral

tegmental area (VTA) which then project into the NAc. In a review of the role of the mesolimbic

pathway in the reinforcement of behavior, Bozarth (1995) describes dopamine as an integral

reinforcer of rewarding behaviors. Behaviors are reinforced through operational conditioning, in DELTA FOS-B MECHANISMS IN NUCLEUS ACCUMBENS AFTER SEXUAL EXPERIENCE Sidibe 6 which a behavior is encouraged or discouraged through a reward or a punishment respectively.

The reward for an appetitive behavior is the activation of the mesolimbic pathway through dopaminergic (and potentially glutamatergic) neuronal transmission.

Current research indicates that sexual behaviors in hamsters, and more generally in rodents, is controlled or facilitated by the mesolimbic dopamine pathway. With the administration of amphetamines into the NAc, a key structure in the mesolimbic pathway, we see an increased facilitation of sexual behavior in male rats as measured by increased copulation frequency, and decreased mounting latency (Fiorino & Phillips, 1999). In addition to the facilitation of sexual behavior, there was an increase in release of dopamine from the NAc after sexual experience, suggesting a role of dopamine in the sexual behavior reward through the mesolimbic pathway. Other works have corroborated the findings that dopamine in the mesolimbic pathway acts as a reward of the behavior, as extracellular levels of dopamine increase in the neurons of the NAc of female Syrian hamsters within 30 minutes of viewing a male hamster (Kolhert, Rowe, & Meisel, 1997). The environment in which the animal is having the sexual experience has been shown to have an effect on mesolimbic pathway innervation as well (Balfour, Yu, & Coolen, 2003). This study had a 2x2 design, with a sexual experience factor

(sexually naïve and sexually experience), and a location of sexual experience factor (in their home cage where they were habituated, and a new cage unassociated with previous sexual experience). Sexual experience paired with an environment similar to the location that they were housed leads an increase in c-fos expression, an indicator of neural activation, in the VTA and the NAc (Balfour et al, 2003). To see increased activation of both the NAc and the VTA after both sexual experience and exposure to sexually-associated environmental cues suggests that DELTA FOS-B MECHANISMS IN NUCLEUS ACCUMBENS AFTER SEXUAL EXPERIENCE Sidibe 7 environmental stability and sexual experience require the activation of the mesolimbic reward pathway.

Inhibiting dopaminergic neuronal activation in key structures of the mesolimbic pathway can also reveal that sexual behavior reward relies on the mesolimbic pathway. In Bradley et al.

(2005b), targeted brain areas of the medial forebrain area, including the NAc, were injected with

6-hydroxydopamine (6-OHDA), a method to target and lesion dopaminergic neurons. Control female hamsters are able to influence the sexual efficiency of the male through movement of their perineum (Bradley et al, 2005a), but in females with medial forebrain dopamine lesions, this ability to facilitate naïve male hamster sexual efficiency, was absent. This suggests that areas in the mesolimbic pathway rely on dopaminergic projections to reinforce sexual behavior and to show the behavioral changes associated with sexual experience. In addition, lesioning mesolimbic pathway brain areas such as the NAc and the VTA using the led to female hamsters who, compared to the controls, had fewer dopamine neurons in each respective brain area when lesioned, and thereby showed less sexual activity (Frye, Petralia, Rhodes, & DeBold 2009).

These lesioned female hamsters also experienced progesterone mediated changes in lordosis durations when compared to controls. This finding of altered lordosis durations, combined with the decreased levels of dopamine in each brain area, suggests and backup previous findings that dopaminergic neurons in mesolimbic structures have a role to play in the facilitation of sexual behavior.

In addition to looking at sexual behavior as a rewarding stimulus, we can look at drug addiction to see mesolimbic activation. The similarities between drug and sex activation of the mesolimbic pathway that allow us to compare these stimuli will be discussed later on in the DELTA FOS-B MECHANISMS IN NUCLEUS ACCUMBENS AFTER SEXUAL EXPERIENCE Sidibe 8 paper. Drug addiction uses the mesolimbic pathway in order to achieve its rewarding effects

(Bozarth 1995). In rodents, many studies indicate that rodents will self-administer drugs by pressing a lever, after being operationally conditioned, in order to attain a reward such as opiates and psychomotor stimulants such as amphetamines or cocaine. This drug intake has been found to activate the same areas that are electrically stimulated to activate the reward system in rats, namely the VTA and the NAc (Bozarth 1995). Lesioning of dopaminergic neurons through 6- hydroxydopamine lesioning (6-OHDA lesioning) was found to reduce drug (amphetamine) seeking behavior through lever pushing (Salamone, Kurth, McCullough, & Sokolowski, 1995).

The reinforcement of the lever pressing behavior with a reward was reinstated 15 minutes after the removal of hydroxydopamine from the system.

Nucleus Accumbens in sexual experience

A common thread throughout the literature on the mesolimbic pathway is the specialized role of the NAc. The NAc is the brain area that receives the most research about the reinforcement of natural and synthetic rewards. As a part of the mesolimbic pathway, it receives dopaminergic projections from the VTA, and then sends its dopaminergic projections out into various other brain regions (Yin, Ostlund, Balleine, 2008). Since the NAc receives these dopaminergic projections, the role that dopamine plays in the NAc is what is thought to make the

NAc important in reinforcement of behaviors.

Observing neurological changes in molecular mechanisms and dopamine in the NAc after a behavioral manipulation shows a clear pattern of upregulation. Dopamine has been found to be upregulated in both male rats (Pleim, Matochik, Barfield, & Auerbach, 1990; Robinson et al,

2001) and female rats (Becker, Rudick, & Jenkins, 2001). In male rats, this can occur at many DELTA FOS-B MECHANISMS IN NUCLEUS ACCUMBENS AFTER SEXUAL EXPERIENCE Sidibe 9

stages of the sexual process. A voltmeter that was placed in a region of dopaminergic neurons

revealed dopaminergic firing during presentation of the female, approach of the male to the

female, and during the actual sexual behavior (Robinson et al, 2001). A behavioral manipulation

known as conditioned place-preference can also lead to an increase in dopaminergic firing in the

NAc. Conditioned-place preference (CPP), is the process through which a rodent associates a pleasurable and rewarding feeling with a stimulus that was associated with previous pleasure

(such as a female or location) (For a review on CPP, see Carboni & Vacca, 2003). Giving both male and female rats a dopamine receptor antagonist (SCH 23390) leads to a disruption of the

CPP induced by cocaine (Nazarian, Russo, Festa, Kraish, & Quinones-Jenab, 2004). CPP has been observed in sexual behavior too, as male hamsters display a CPP towards both location of sexual experience, and chemo-sensation of pheromones release by the female (Bell, Meerts, &

Sisk, 2010), presumably, although not definitively, due to an increase in dopamine in the NAc.

In addition to manipulating behavior to see neurological changes, one can directly

innervate the NAc in order to reveal its role in sexual behavior reward. Using the CPP paradigm,

hamsters that are given dopamine antagonists show a marginally reduced preference for the

location of sexual experience than controls that receive a vehicle injection (Carbonioppa, &

Rowe, 1996). In addition, many instances of manipulation of the NAc through 6-OHDA have

revealed that a decrease in dopaminergic NAc neurons leads to a decrease in the reward of

behaviors as measured through decreased sexual behavior activity (Bradley et al., 2005ab; Frye

et al, 2009). Optogenetics, a method in which one can create light sensitive neurons to selectively

control neuronal activation by adjusting the level of light, was used to find if addiction behavior

could be affected by turning off and on the neurons in the NAc (Stefanik, Kupchik, & Kalivas, DELTA FOS-B MECHANISMS IN NUCLEUS ACCUMBENS AFTER SEXUAL EXPERIENCE Sidibe 10

2014). The researchers then trained the rats for a typical lever pressing task, and promptly extinguished the behavior through light inhibition. When trying to reinstate the behavior after inhibiting both afferent and efferent neurons, it was found that the behavior was unable to be reinstated. This suggests that the NAc has a role in the creation, and the reinstatement of a rewarding behavior (in this case, cocaine seeking behaviors).

A more in-depth look into a specific anatomical feature of the NAc is important in understanding the rewarding effects of sexual behavior with more specificity. In literature, a distinction is made between two parts of the NAc: the core and the shell. There are some morphological differences between the shell and the core that are important to define, as manipulations in either area can have different implications in the interpretation of the findings.

The various experiments performed by Meredith, Ypma, and Zahm (1995) highlight the distinct neuroanatomical differences between the shell and the core of the NAc. The neurons in the shell project to the ventral pallidum, extended amygdala, lateral hypothalamus and the ventral tegmentum, while neurons in the core extend to dorsolateral ventral pallidum, sub-thalamic nucleus, and the substantia nigra structure. Lesions to the core and shell of the NAc of rats who were administered cocaine on a continuous schedule revealed that lesions to the core, rather than to the shell, attenuated drug seeking behaviors, as measured by the rate of lever pushing (Ito,

Robbins, & Everitt 2004). Lesions to the shell seemed to have less or no effect on drug administering behaviors. However, a specific subset of neurons in the shell of the NAc in male rats were found to fire more during copulatory behaviors such as mounting, intromission, and ejaculation (Matsumoto, Urakawa, & Hori 2012), opposing the results of Ito et al. (2004). DELTA FOS-B MECHANISMS IN NUCLEUS ACCUMBENS AFTER SEXUAL EXPERIENCE Sidibe 11

However, since Matsumoto et al. (2012) did not test how the NAc core affects sexual behavior, the results of this study are not conclusive.

Prior and current research suggests that the NAc has been reliably found to correlate with rewarding behaviors. However, no mechanism has been discussed to suggest the reinforcement of rewarding behaviors such as sexual behavior. The reinforcement of sexual behavior has also been linked non-causally to dopaminergic levels in the NAc. Meisel & Mullins (2006) discuss this reinforcing effect of dopamine on sexual behavior. Previously sexually naïve hamsters naturally produce a small increase in extracellular dopamine in the NAc after their first sexual encounter, while hamsters that are already sexually experienced hamsters receive a larger increase of dopamine after an individual experience. Kohlert & Meisel (1999) performed the used a more detailed time-scale using 3 groups of hamsters, those who had 6 weeks of sexual experience, those that had 3 weeks of sexual experience, and those that were sexually naïve. As in Meisel and Mullins (2006), the female hamsters that had the most sexual experience were produced more dopamine in the NAc after an additional sexual experience, compared to the other two groups. The results of both studies suggest that there is a mechanism in place that allows for more dopamine to be released in the NAc in more sexually experienced hamsters, suggesting that there is some mechanism that leads to the sensitization and reinforcement of sexual behavior. This type of change, as will describe next, turns out to be a structural change in the neurons in the NAc. More specifically, this is an increase in the density of dendritic spines.

Dendritic Spine complexity and sexual experience –

Dendritic spine growth is an important function in the learning of behaviors and reinforcement. Dendritic spine changes occur in mostly all neurons that change and grow as a DELTA FOS-B MECHANISMS IN NUCLEUS ACCUMBENS AFTER SEXUAL EXPERIENCE Sidibe 12

result of an experience- dependent stimulus. A prominent area in research that includes dendritic

spine growth and how it’s important in reinforcement, comes from the field of learning and memory. The process of increased dendritic spine growth and the resulting increase in sensitization of the synapse is known as long term potentiation (LTP). LTP, first introduced by

Hebb (1949), is the process where after repeated activation, neurons become quicker to activate, and therefore create a strong response. This is due to an increase of a certain type of glutamatergic receptor known as an AMPA receptor. This increase in activation of AMPA receptors dislodges magnesium ions from NMDA receptors, allowing calcium to flow in once glutamate binds to it. The calcium ions act as a second messenger to recruit more AMPA receptors and activate neuronal growth factors to increase the number of dendritic spines. This gives neurotransmitters an increased surface area to activate the neuron, allowing for a stronger response of the neuron. A stronger response means that a stimulus that would normally marginally activate a neuron, would activate the same neuron more strongly after LTP occurs.

On a large scale, increasing the likelihood of activation of numerous neurons in a certain brain area leads to this stronger activation of the brain area.

Evidence from the literature supports the idea that increased dendritic spine density is important in the process of LTP. After the encoding of a memory, there is an increase in the number of short dendritic spines in the hippocampal neurons (Kasai et al., 2004). These dendritic spines come with an increased number of AMPA receptors on them, leading to more places for neurotransmitters to activate the neuron, and thus more neuronal firing. Inducing LTP by stimulating individual neurons, leads to an increase in AMPA receptors, as well as an increase in dendritic spine size and density (Yuste & Bonhoeffer, 2001). Furthermore, increasing both the DELTA FOS-B MECHANISMS IN NUCLEUS ACCUMBENS AFTER SEXUAL EXPERIENCE Sidibe 13 number of receptors and dendritic spine density allowed for overall increased levels of synaptic activity. A similar test of stimulating various neurons in the rat for a duration of one hour revealed an increase in LTP, and found that the number of dendritic spines increased dramatically in these areas (Toni, Buchs, Nikonenko, Bron, & Muller, 1999). Additionally, pharmacologically blocking a protein important that is important in synaptic development halted the ability for the hippocampal neurons to induce LTP, due to the lack of increased dendritic spine density and AMPA receptor increase. This correlation of dendritic spine growth to increased LTP in hippocampal cells proposes the importance of physical changes of the neuron for the development of LTP.

Evidence of LTP in the NAc is found in an important morphological feature of the NAc known as the medium spiny neuron (MSN). MSNs are found to have an increased density of dendritic spines as compared to other neurons (Shen, Sesack, Toda, & Kalivas), and are found to be the primary type of neuron in the NAc (Li, Kolb, & Robinson, 2003; Staffend, Hedges,

Chemel, Watts, & Meisel, 2013; Song et al., 2014). The higher amount of dendritic spines in the

MSNs in the NAc is indicative of stronger synaptic connections. In addition to having many more dendritic spines, and therefore dopaminergic synapses, the importance of the MSN in the

NAc for reinforcement lies also in the type of dopamine receptors that it expresses. There are many subtypes of dopamine receptors, but the two that are expressed in the NAc are D1 and D2

(Hopf, Cascini, Gordon, Diamond, Bonci, 2003). D1 and D2 receptors have different functions in the process of neurotransmission, and have been implicated in learning and memory (Manago,

Castellano, Oliverio, Mele, & Leonibus, 2008), but have both been implicated in the reward of sexual behavior (reference?). In fact, while D1 receptors are found to enhance synaptic DELTA FOS-B MECHANISMS IN NUCLEUS ACCUMBENS AFTER SEXUAL EXPERIENCE Sidibe 14 excitatory activity in the NAc, the D2 receptors are found to decrease the level of synaptic activity in MSNs (Surmeier, Ding, Day, Wang, & Shen, 2007; Podda, Riccardi, D’Asenzo,

Azzena, & Grassi, 2010).

Observing the effects of sexual experience on dopamine receptor subtype expression can elucidate the role of D1 and D2 receptors in MSNs of the NAc in reinforcing sexual behavior.

Hamsters that were seuxally experienced had an increase in dendritic spine density of the MSN in the core of the NAc and an increase in mature spine morphological characteristics (called filopodial dendrites) (Staffend et al, 2013). Secondly, MSNs that express D1 receptors were upregulated in the core in sexually experienced hamsters compared to sexually naïve hamsters.

The number of D2 receptor-expressing MSNs were not significantly different in the NAc core compared to controls. In addition, blocking D1-receptor involvement by using the D-1 receptor antagonist (SCH23390) led to a decrease in the number of mounts and intromissions, and increased the post-ejaculatory interval (Ahlenius & Larsson, 1990). Conversely, giving a rat a dopamine agonist was sufficient for seeing upregulation of cAMP in the NAc of sexually naive hamsters (Bradley et al, 2004). Since cAMP is a second-messenger system that is used in the process of LTP, an increase in cAMP would indicate that D1 receptors mediate a process of sexual behavior reinforcement. Since D1 receptors are found to be involved in the learning and reinforcement of a behavior, and are upregulated in MSNs of the NAc after sexual experience, this could point towards the role of the MSNs in the NAc in the learning and reinforcement of sexual behavior.

DELTA FOS-B MECHANISMS IN NUCLEUS ACCUMBENS AFTER SEXUAL EXPERIENCE Sidibe 15

Using drug studies as evidence of sexual behavior reinforcement

An issue with the current literature on upregulation dendritic spine density in the NAc is

that it mostly uses drug models rather than sexual behavior models. Few studies having looked

exclusively at the growth of dendritic spines in the NAc as it relates to sexual experience

(Pitchers, Vialou, & Nestler, 2013). Therefore, many of the studies that will be mentioned focus

on drug rewarding behaviors. However, this does not suggest that drug reward is more important

than sexual reward. This merely suggests that research has been focused around drug

reinforcement, perhaps because it is an easier model to study.

There is a way that drug reward findings can be generalized to sexual behavior research

findings. This is through the idea of behavioral and cross-sensitization. Behavioral sensitization

is the process through which an animal becomes more behaviorally responsive to the same

stimulus after drug-induced morphological changes in the brain. Drug-induced increases of

dendritic density of neurons in the NAc is reliably correlated with behavioral sensitization, as

drug administration paradigms have been found to reliably co-induce sensitized drug seeking

behavior and increases in dendritic spines and branching (Russo et al., 2010). Other studies have

corroborated this report, as rats that self-administer amphetamines show more dendritic spine growth on MSNs in the NAc compared to those who were manually given amphetamines or sugar (Robinson, Gorny, Mitton, & Kolb, 2001). These studies suggest that dendritic spine growth in the NAc is important in reinforcing drug seeking behavior.

Cross-sensitization is the process through which the morphological changes in dendritic spines we see from drug use causes an increased responsiveness of sexual behaviors. The reason that cross-sensitization is important is because if cross-sensitization is observed, it is possible DELTA FOS-B MECHANISMS IN NUCLEUS ACCUMBENS AFTER SEXUAL EXPERIENCE Sidibe 16

that both the drug reinforcement pathway and sexual reinforcement pathway use similar

mechanisms for reinforcement. At the least, cross sensitization shows that the increase in growth

of dendritic spines in the NAc is used as a way to reinforce both drug behaviors and sexual

behaviors. Evidence of cross-sensitization of drug reinforcement to sexual reinforcement has

been observed in many cases. Treatment of sexually naïve male rats with d-amphetamine led to a decreased mount and intromission latency compared to control rats who didn’t receive d- amphetamine (Fiorino & Phillips, 1999). Additionally, consistent administration of d- amphetamine in sexually naïve female hamsters leads to more sexually appetitive behaviors

(psychomotor increases, solicitations, hops and darts, etc.) compared to hamsters that are given saline (Afonso, Mueller, Stewart, & Pfaus 2009). This finding suggests that d-amphetamine administration is sufficient to increase sexualy appetitive behaviors in rats, which indicates that cross sensitization has occurred.

Cross-sensitization can also increase the density of dendritic spines in addition to behavioral sensitization. Injecting amphetamines into the NAc leads to increased sensitization of locomotor responses in sexual behavior as a result of an increase of dendritic spine density in rats

(Pitchers et al, 2010). When given d-amphetamine, sexually experienced female hamsters have higher levels of c-fos expression, a marker of increased neuronal activity, in the NAc core compared to sexually naïve female hamsters (Bradley & Meisel, 2001). In addition, while both sexually experienced and naive hamsters responded to the amphetamine by showing increased

general locomotor activity during sex, the sexually experienced group responded quicker and

stronger. This evidence of cross-sensitization suggests that sexual behavior uses the same

pathway for its effects as drug taking behavior. Lastly, amphetamine dependent increases in DELTA FOS-B MECHANISMS IN NUCLEUS ACCUMBENS AFTER SEXUAL EXPERIENCE Sidibe 17 dendritic spine density resulted in changes in locomotor activity after injecting amphetamines directly into the NAc. This increase in dendritic spine density was then correlated with sexual experience, such that amphetamine-induced dendritic spine density increases were greater in those with more sexual experience (Pitchers et al., 2010b)

With the link between drug-induced neuroplasticity and sexual behavior sensitization established, we can apply the findings of drug reinforcement and increased dendritic spine density to sexual behavior reinforcement. Many types of drugs have been implicated in dendritic spine density of the NAc. Morphine has been found to alter the density of dendritic spines in the

NAc of rodents trained to self-administer the drug as opposed to those rats that were merely given the drug (Russo et al, 2010). Introducing amphetamines, a psychomotor stimulant, into the

NAc leads to an increase in dendritic spine density of distal dendrites (which are the dendrites that actually form synapses with other neurons) in both the NAc (Robinson & Kolb, 1999; Li et al, 2003) and the caudate (Li et al., 2003). Cocaine, another psychomotor stimulant, has been implicated in dendritic spine density (Russo et al, 2011). Inhibiting the effects of cocaine using cyclin-dependent kinase 5 (cdk5), a protein involved in mediating the effects of drugs using dopaminergic pathways, leads to a direct decrease of dendritic spine density (Bibb, 2003;

Norrholm, Bibb, Nestler & Ouiment, 2003). In this case, interestingly, the decrease of dendritic spines after inhibiting cdk5 was found in the shell and not the core. Additionally, changes in dendritic spine density of the NAc after cocaine administration relies on the inhibition of dopamine transporter proteins, emphasizing the role of dopamine in drug induced synaptic plasticity changes (Martin et al, 2011). All these drug studies share in common that administering a drug to the NAc will result in an increase in dendritic spines. Since the process DELTA FOS-B MECHANISMS IN NUCLEUS ACCUMBENS AFTER SEXUAL EXPERIENCE Sidibe 18 of cross-sensitization is well documented, the increased density of dendritic spines seen after drug administration could hint at a role in sexual behavior reinforcement.

Despite the plethora of evidence suggesting the role of increased dendritic spine density in sexual behavior reinforcement, one question still remains. It has been established that sexual experience leads to activation of the mesolimbic pathway, which in turn increases the level of dopamine in the NAc. There is, however, no evidence to suggest that dopamine directly increases the density of dendritic spines, but that it merely activates the increased receptors on the dendritic spines. In other words, dopamine increase is not directly responsible for the effects of long term potentiation of neurons in the NAc. Therefore, there must be some sort of mechanism in place that leads from increased activation of neurons in the NAc to the increase of density of dendritic spines. What molecular mechanisms lead to the growth of dendritic spines, and thereby play a role in the reinforcement of sexual behavior?

Targeted Molecular Mechanism Involved in Increased Dendritic Spine Density –

Molecular pathways exist for many neural processes, and many of them are complex and require many biological interactions. There is a full-fledged molecular pathway for the upregulation dendritic spines, involving actin cytoskeleton proteins, and multiple signaling pathways involving different types of kinases, scaffolding proteins, and surface receptors (Tada

& Sheng, 2013). However, we were only able to look at one particular protein, given the time constraints. The molecular mechanism that was chosen, that has been particularly involved in dendritic spine growth, is known as delta fos-b. Delta fos-b is part of a family of transcription factors which includes c-Fos, a gene that is used as a marker for the expression of other many other genes (Nestler, 2008). Transcription factors are proteins that can bind to specific regions DELTA FOS-B MECHANISMS IN NUCLEUS ACCUMBENS AFTER SEXUAL EXPERIENCE Sidibe 19 that occur before a gene and regulate its expression (how much of the protein is made and when).

The binding of all transcription factors in this regulatory module is necessary for transcription to occur effectively, which is why transcription factors can play such a large role in regulation of gene expression. Delta fos-b is an alternative splice of the fosB gene, meaning that fosB gene is transcribed in many different ways before it is translated into a protein (Nestler, Barrot & Self,

2001).

Like in research on dendritic spines and mesolimbic pathway, delta fos-b and its role in drug reinforcement receives more attention than sexual behavior reinforcement. However, evidence that this same cross-sensitization between sexual reward and drug reward found at the level of the dendritic spines is present at the level of delta fos-b. Suppressing the transcriptional activity of delta jun-d (therefore breaking up the heterodimer) impairs the sensitization of behaviors to drug intake in sexually experienced rats, specifically with regard to amphetamines

(Nestler et al, 2001). In addition, delta fos-b induced through sexual experience leads to sensitization of the behavioral response to amphetamines (Beloate, Weems, Casey, Webb, &

Coolen, 2015). Interestingly, this sensitization seemed to be mediated by the glutamate NMDA receptor. This list of studies suggests a cross-sensitization of addicting drugs to sexual behavior which in turn suggests that sexual reward and drug reward behaviors use similar molecular pathways.

It is important to establish two things in order to show delta fos-b as a reliable factor in the reinforcement of sexual behavior. First, expression levels of delta fos-b must be higher in the

NAc after sexual experience compared to the sexually naive group. Secondly, delta fos-b must be shown to increase dendritic spine density in neurons in the NAc, especially in the NAc core. DELTA FOS-B MECHANISMS IN NUCLEUS ACCUMBENS AFTER SEXUAL EXPERIENCE Sidibe 20

These two facts are important because if delta fos-b plays a role in the reinforcement of sexual behavior, it must activate the NAc due to the role of the NAc in sexual behavior, and increase dendritic spine density due to the role of dendritic spines in reinforcement.

Sexual behavior has been reliably found to increase, and be increased by, the expression of delta fos-b. Delta fos-b overexpression and downregulation (lowering the expression of genes), can be used to test sexual behavior reinforcement paradigms (Nestler, 2008).

Overexpressing delta fos-b leads to increased levels of sexual reward, shown by increased time spent in the chamber where the sexual experience occurred (Hedges, Chakravarty, Nestler, &

Meisel, 2009). In addition, overexpression of delta fos-b led to increased efficiency of copulatory behavior in female hamsters paired with naïve males, as measured by the increased hit and intromission rate of the males. The same is found in sexually experienced males, as increased levels of delta-fos b in areas of the mesolimbic pathway, including the NAc, was present in sexually experienced hamsters more than in sexually naïve hamsters (Pitchers et al, 2010). In a subsequent experiment, they found that the upregulation of delta fos-b in the NAc mediates the reinforcement of several sexual behaviors such as intromission and ejaculation, decreasing their latencies. Furthermore, overexpressing levels of delta jun-d (a negative heterodimer of delta fos- b) by viral vector in the NAc core of female hamsters leads to a decrease in conditioned place preference in sexually experienced hamsters (Been, Hedges, Vialou, Nestler, Meisel, 2013).

Lastly, one study searched the effect of naturally rewarding behaviors on levels of delta fos-b in the NAc. What they found was that sucrose seeking and increased sexual behavior led to an increase in delta fos-b in the NAc (Wallace et al., 2008). In addition to behavior affecting level of expression, they also found that increasing the level of expression of delta fos-b led to DELTA FOS-B MECHANISMS IN NUCLEUS ACCUMBENS AFTER SEXUAL EXPERIENCE Sidibe 21 behavioral sensitization of sexual behavior, shown though increased performance of sexual behaviors. This set of studies strongly implicates delta fos-b in sexual reward through its effect on the NAc.

Although delta fos-b expression is reliably upregulated in the NAc after sexual experience, it is important to show that this expression is co-increased with dendritic spine density. The fact that delta fos-b is a transcription factor is important, because regulating expression of a gene changes the amount of protein product. If a protein product plays an important role in building dendritic spines and is downregulated, the efficacy of forming dendritic spines will decrease. Therefore, upregulating it may increase the amount of dendritic spine growth. After introducing a stimulant drug known as methylphenidate, it is possible to co- locate the increase in expression of delta fos-b and the increase in short and long growth of dendritic spines in the core of the NAc (Kim et al, 2008). Furthermore, by increasing the level of delta fos-b in the NAc of hamsters, there is a subsequent increase in dendritic spine growth, specifically in the medium spiny neurons that contain D1 receptors (Grueter, Robison, Neve,

Nestler, & Malenka, 2013). This growth was only found significantly in the less mature dendritic spines (such as mushroom spines), meaning that this wasn’t a widespread increase. Overall, however, there seems to be evidence to link dendritic spine growth and delta fos-b expression in the NAc.

Hypotheses and predicted results-

Sexual behavior hypotheses – DELTA FOS-B MECHANISMS IN NUCLEUS ACCUMBENS AFTER SEXUAL EXPERIENCE Sidibe 22

Since all hamsters in the group will be sexually naive on week 0, we expect that the hamsters will show evidence of sexual inefficiency as marked by a low intromission to ejaculation rate. However, as sexual experience increase, we expect that the hamsters will become more efficient, and show a much higher intromission to ejaculation rate. Our sexual behavior hypothesis is as follows:

1. There will be a significant increase in sexual efficiency (intromissions divided

ejaculations) over the six weeks of sexual experience as measured by decreased

intromissions needed to achieve ejaculation, and fewer mounts needed for a successful

intromission.

2. There will be a significant decrease in lordosis latency, and an increase in lordosis

duration across sexual experiences.

Delta fos-b hypotheses -

Given the strong evidence found on delta fos b and the role it plays in the reinforcement of sexual behavior, we believe that delta fos-b will influence neural plasticity in the NAc following sexual experience. From this, we came up with the following hypothesis:

1. If delta fos-b plays a critical role in the molecular pathway underlying neuroplasticity in

the brain, then there should be an upregulation of delta fos-b in the NAc of sexually

experienced hamsters over sexually naïve hamsters.

This hypothesis comes from the research that sexual experience leads to an upregulation of delta fos-b (Nestler, 2008; Hedges et al., 2009; Pitchers et al., 2013). DELTA FOS-B MECHANISMS IN NUCLEUS ACCUMBENS AFTER SEXUAL EXPERIENCE Sidibe 23

In addition to the finding of delta fos-b in the NAc, we are hoping to add to the scientific community by looking at the potential for sex differences in the reinforcement of sexual behavior through delta fos-b. In other words, do male hamsters have a different molecular pathway or mechanism in order to increase the density of dendritic spines in the NAc?

Unfortunately, there has not been much research performed on the differences between the molecular mechanisms underlying dendritic spine growth in the NAc after sexual experience.

After a comprehensive literature search, there was one article that tested sex differences of delta fos-b. By comparing the analgesic effects of morphine on wild-type FosB mice against mice with null mutations of the gene, they found that the analgesic effects of morphine were mediated by delta fos-b in female mice but not in male mice (Solecki et al., 2008). However, we cannot make conclusions on this piece of evidence. First of all, this is the only finding relating to sex differences. Another problem is that although cross-sensitization of sexual reward and drug reward allows us to compare them closely, this study looks at analgesic effects instead of drug reward behavior (such as pushing a lever for more of the drug). Since these are not the same processes, we cannot infer that drug analgesia is a sign of the difference between drug rewards between sexes mediated by delta fos-b.

Since there is not enough research in sex differences between male and female hamster molecular mechanisms in reinforcing sexual behavior, we cannot postulate whether there are sex differences or not. The only evidence that there could be some molecular differences involved is through the fact that male and female hamsters have different roles in sexual behavior (Siegel,

1985; Hull & Dominguez, 2007). This could potentially lead to different molecular mechanisms of sexual reinforcement, but we no evidence to suggest whether this difference in sexual DELTA FOS-B MECHANISMS IN NUCLEUS ACCUMBENS AFTER SEXUAL EXPERIENCE Sidibe 24

behavior applies to different mechanism of increasing dendritic spine density and therefore

reinforcement of sexual behavior. Therefore, our second hypothesis is as follows:

2. If the reward pathways in male and females are the same, then we should not find a

difference in the upregulation of delta fos-b in the NAc between males and females.

Methods:

Subjects –

We used Syrian (Golden) hamsters from Charles River Laboratory breeders (Wilmington,

MA). There was a total of 24 hamsters, with 12 being male and 12 being female. Within each sex, 6 will be control subjects while the other 6 will experimental subjects, and will receive

sexual experience. The reasoning for 6 hamsters in each group is due to previous research

showing that 6 hamsters provide us with enough statistical power to detect significant differences

(Bradley & Meisel, 2001; Bradley et al., 2005; Pitchers et al., 2010) while also limiting the

number of hamsters needed for sacrifice. Wild Syrian hamsters are nocturnal, meaning that they

sleep during the day and are awake at night. For convenience sake, they are housed in the

laboratory on a reverse light-dark cycle so we can observe them during the day. All hamsters

were housed individually in order to eliminate confounds created through pairing either males

together, or females together. Aspen bedding, wooden blocks, and plastic tubes were provided

for the comfort and entertainment of the hamsters. Food and water were given to the hamsters ad

libitum. Ambient temperatures were maintained between 68 and 72 degrees Fahrenheit. All DELTA FOS-B MECHANISMS IN NUCLEUS ACCUMBENS AFTER SEXUAL EXPERIENCE Sidibe 25

procedures were in accordance with the IACUC protocol and the National Institute of Health

guidelines for the care and use of animals.

Procedure -

Ovariectomy and Hormone Priming –

All female hamsters, regardless of which experimental group they were in, received an

ovariectomy. This ensured that they will not become pregnant during the behavioral testing and

also allow us to control levels of ovarian hormones and therefore control when the females are

sexually receptive. Hormones were dissolved in cotton seed oil and injected subcutaneously in

order to facilitate the transfer into the body. Females were hormonally primed with estradiol benzoate (0.1 micrograms per 0.1 ml of cottonseed oil) both 48 hours and 24 hours before a sexual experience, and progesterone (100 micrograms per 0.1 milliliter) 4 hours before each sexual encounter. This allowed the female to show receptive behaviors, such as lordosis when it is placed with a male hamster. Both control and experimental groups received hormone primes in order to control for the effects of each hormone on brain changes. Since male testosterone release is persistent over time, and only minimal doses are required for male specific behaviors

(Damassa, Smith, Tennent, & Davidson, 1977; Piekarski et al, 2009), the testes will not be removed from the male and thus they will not be hormonally primed.

Sexual behavior testing –

After hormone priming, the hamsters received sexual experience. Both females and males

in the sexually experienced group had 6 weeks of sexual experience, with one encounter

occurring each week. Each week, a new male was placed with a new female in order to avoid DELTA FOS-B MECHANISMS IN NUCLEUS ACCUMBENS AFTER SEXUAL EXPERIENCE Sidibe 26 any individual mating preference effects. In the meantime, the sexual naïve female group continued to receive hormone priming at the same time as the sexually experienced hamsters.

Four hours after priming female hamsters with progesterone, the experimental group of male hamsters were placed in the cage with the female. In order to allow the male hamster an ample amount of time to display each category of sexual behavior, each sexual experience lasted for 10 minutes. After the 10 minutes are through, the male hamsters will be removed, and put in their own cages until their next sexual encounter a week later.

The following behaviors were monitored: For males, the amount of mounting, intromission, and ejaculations will be measured. For females, lordosis latency (how long it takes to achieve lordosis) and lordosis duration (total time spent in lordosis) will be measured.

Hamsters that received significantly different levels of sexual experience that could lead to a difference in the amount of delta fos-B expression were taken out of consideration for only the experience. Outliers were determined through taking an average of the data for sexual experience and eliminating subjects from only the experience in which their recorded behavioral measure data was more than 2 standard deviations outside the mean. Especially concerning are those that have little sexual experience, as it would show that the hormone priming was ineffective or otherwise deficient and skew expression levels of delta fos-b towards sexually naïve hamsters.

Each sexual encounter was video recorded in order to precisely observe and count evidence of each sexual measure.

Anesthetization and Sacrifice of Hamsters –

After 6 weeks of sexual experience in the experimental group, and after 6 weeks of no sexual experience for the control group, each hamster was given an overdose of sodium DELTA FOS-B MECHANISMS IN NUCLEUS ACCUMBENS AFTER SEXUAL EXPERIENCE Sidibe 27 pentobarbital and underwent anesthetization before removal of the brain. The anesthetization performed to make them unconscious during the decapitation process. The process of sacrifice occurred through rapid anesthetized decapitation following by the removal of the brain. The removal of the brain occurred imminently after the death of the hamster in order to preserve brain activity.

Collecting samples –

Once the brain was removed, it was placed into a brain matrix to section the brain.

Chilled razor blades were used to section the brain. 2 mm tissue punches were of both sides of the brain of NAc and CP and were and put into a test tube. Test tubes with the brain material were then quickly sprayed with freeze spray and put into a container of dry ice to avoid time- dependent decreases of delta fos-b expression. The CP, part of the dorsal striatum, will act as a control area because it is an adjacent area of the brain, and part of the same general structure, but not thought express delta fos-b in response to sexual behavior.

Protein Assay and Western Blotting -

After processing the tissue, we homogenized and assayed the tissue. Protein sample amounts were determined using the Bradford Protein Assay method. Samples were then prepared, inserting the same amount of Laemelli buffer into a tube as protein sample. After preparation, samples were inserted into the gels. A current was then run through the gel in order to get protein samples placed in the wells to move from the top of the gel to the bottom. Those protein products that are smaller travel faster because they can filter through the gel easier, while the bigger protein particles will move slower. A ladder was placed into one of the wells, which DELTA FOS-B MECHANISMS IN NUCLEUS ACCUMBENS AFTER SEXUAL EXPERIENCE Sidibe 28

served as a standard in which to compare our bands. The current ran for one hour, by which time

the particles will have travelled as far as possible before getting stuck.

After the gel was run, it was then transferred onto a nitrocellulose membrane by placing the membrane onto the gel and sending a current through them. Antibodies generated against delta fos-b were used to visualize delta fos-b protein levels between experimental conditions.

After 5 washes of 5 minutes each, the secondary antibody was attached. Both antibodies were necessary to visualize the bands on the scanner. After secondary antibodies, membranes were scanned using a spectroscopy machine. The denser the lines are on the nitrocellulose paper, the more protein that was found in that from that specific well. We used an optical density measure to quantify the differences in the expression of protein between each condition. Optical density was found by the measurement tool on the spectroscopy reader. By selecting the area of each band on the gel, the computer gave us a reading for the density of the line.

In order to control for differences in how much protein was added to each well, we also blotted each sample for GAPDH, a protein that is found in high quantities, in addition to delta

fos-b. GAPDH protein is present in all cells and levels do not change in response to behavior,

making it an excellent loading control (Barber, Harmer, Coleman, & Clark, 2005). After running the blots with both delta fos-b and GAPDH antibodies, we divided the delta fos-b reading by the

GAPDH reading to get a mean adjusted value.

Statistical analyses -

We used a one-way ANOVA to look for a significant difference between adjusted delta fos-b levels between our sex experienced and sex naïve groups, while a separate one-way DELTA FOS-B MECHANISMS IN NUCLEUS ACCUMBENS AFTER SEXUAL EXPERIENCE Sidibe 29

ANOVA to find the differences between expression levels of delta fos-b in males against females

will satisfy the second hypothesis. A repeated measures ANOVA was run to test for an effect of time on the improvement of efficiency of sexual behavior. Each behavior was compared across experience for both males and females. All statistical tests will be run using the statistical program SPSS.

Results:

Sexual behavior analysis

We ran a repeated measures ANOVA to test for significant differences across sexual

experience in the following sexual behavior categories: lordosis latency, lordosis duration,

number of mounts, intromissions, ejaculations, and a measure of sexual efficiency (intromissions

divided by ejaculations). The between-subjects factor was experience number, and the within-

subjects factor was the sexual behavior measure. After six weeks of sexual experience, lordosis

latency did not significantly decrease across repeated experiences (F(5,5) = 2.17, p > .05).

Similarly, lordosis duration did not significantly increase across repeated experiences (F(5,5) =

1.70, p > .05). Post hoc tests were not performed due to a lack of significance in our omnibus repeated measures ANOVA. For males, mounts (F(5,5) = 0.10, p > .05), intromissions (F(5,5) =

0.32, p > .05), and ejaculations (F(5,5) = 2.01, p > .05) did not significantly change across repeated sexual experiences. For both females and males, we failed to reject the null hypothesis.

Delta fos-b pilot experiment DELTA FOS-B MECHANISMS IN NUCLEUS ACCUMBENS AFTER SEXUAL EXPERIENCE Sidibe 30

To practice our western blot techniques, we ran a pilot experiment with 4 sexually naïve

male hamsters involving the same procedure as our main experiment. Delta fos-b expression was

evident across all of our subjects. This signified that our procedure was able to produce results.

The positive results were the impetus for our subsequent tests during the troubleshooting period

(discussed later).

Expression of Delta Fos-B between sexual experience groups We found that sexually experienced females had significantly increased levels of delta

fos-b expression in the NAc compared to naive females (F(1,10) = 21.43, p = .001). In the CP

control brain area, where delta fos-b expression should not change across sex experience groups,

there was no significant difference between female sexually naive hamsters and sexually

experienced hamsters in the NAc (F(1,10) = 0.851, p > .05).

We found that sexually experienced males also had significantly increased levels of delta fos-b expression in the NAc compared to naïve males (F(1,2) = 25.20, p < .05). As was the case in females, there was no difference between sexually experienced males and naïve males in the

CP (F(1,6) = .975, p > .05).

Expression of Delta fos-b between Males and females

Our second hypothesis stated that there would be a difference in delta fos-b expression in

the NAc between the male sexually experienced group and the female sexually experienced

group. There was no difference in delta fos-b expression levels between males and females in the

NAc (F = 1.51, p > .05). To serve as a control, we compared delta fos-b levels of expression in

the CP. It was again found that there was no significant difference between males and females (F DELTA FOS-B MECHANISMS IN NUCLEUS ACCUMBENS AFTER SEXUAL EXPERIENCE Sidibe 31

= 0.107, p > .05).In naïve hamsters, there was no difference in expression of delta fos-b between males and females in either the NAc (F(1,6) = 2.706, p > .05) or the CP (F(1,7) = 1.326, p >

.05).

Discussion:

Troubleshooting lack of Delta fos-b results -

After running multiple gels and using primary and secondary antibodies for delta-fos b,

we were unable to detect bands in the imaging phase of the Western Blot. With no detection of

delta fos-b, we began troubleshooting for the issue that led to our results. We first attempted to

solve the problem by increasing the amount of protein that was run by 4-fold. We felt that this

was enough to ensure that if no protein were discovered, that there was no delta fos-b in our

sample. After the running 2 of our gels again, and attaching primary and secondary antibodies,

there were no bands of delta fos-b. From this finding, we concluded that there could have been

an issue with tissue collection and homogenization, or that there could have been a problem with

the transfer buffer. In order to discover the problem, we re-blocked two of our gels, and attached

primary and secondary antibodies of the GAPDH protein. After running the gel with quadruple

our previous sample with GAPDH antibodies, we found evidence of banding when imaging.

This would suggest that there is some protein in our samples, and that delta fos-b is present to a

degree, but is not being picked up by our current level of protein or concentration of antibodies.

Next, we wanted to see if the problem was that there were small amounts of delta fos-b in

our sample. In order to test this, we decided to image our membranes with the quadrupled protein DELTA FOS-B MECHANISMS IN NUCLEUS ACCUMBENS AFTER SEXUAL EXPERIENCE Sidibe 32 sample, along with a 1:300 blocking to antibody concentration instead of the typical 1:1000 antibody concentration. When imaging the blots, we found a strong signal of delta fos-b. This was the first evidence of any delta fos-b in our non-pilot study. From these tests we surmised that high concentrations of both primary antibody and protein were necessary to find any evidence of delta fos-b. In order to be more thorough in our analysis, we proceeded to run our last 4 gels with high concentrations of antibody and protein. When we ran the analysis, we found that all gels showed evidence of delta fos-b. We ran the same gels with the GAPDH to serve as a control for the delta fos-b. This result confirmed to us that quadrupling the amount of protein and tripling the concentration of antibody was necessary to get results.

Discussion of Results -

Sexual behavior findings -

Our sexual behavior hypotheses were not supported by the data. It is possible that the lack of sexual efficiency of our hamsters is because hamsters are unable to become sexually efficient. However, evidence has shown that this may not be the case. There is an observable increase in copulatory efficiency in sexually experienced rats (Hull & Dominguez, 2007).

However, this increase in efficiency was only found after multiple periods of 7-8 ejaculations per sex experience. In another instance, rats that were given 20 minutes of sexual experience daily also showed signs of copulatory efficiency, evidenced by shorter post-ejaculatory intervals

(Freidin & Mustaca, 2004). Our hamsters frequently had 0 or 1 ejaculations in each sexual experience, and were only given 10 minutes to mate, for six weeks. Therefore, it is more likely that the lack of an increase in sexual efficiency in our hamsters was due to a problem in experimental design. It is likely that the amount of sexual activity is in our study was too DELTA FOS-B MECHANISMS IN NUCLEUS ACCUMBENS AFTER SEXUAL EXPERIENCE Sidibe 33

infrequent to increase efficiency in the hamsters. In addition, the small number of frequencies

also likely impacted the ability for the male hamster to become more efficient. An exhaustive search of the literature led to no studies that investigated sexual efficiency in female rodents.

However, it may be the case that having more time to mate, as well as mating more frequently, would lead to an increase in sexual efficiency.

Delta Fos-b findings -

We were able to corroborate previous data that showed increases of delta fos-b

expression in the NAc after 6 weeks of sexual experience in rodents (Wallace et al, 2008;

Hedges et al, 2009; Been et al, 2013; Pitchers et al, 2013). This supported our hypothesis, as we

predicted that that would be a difference in delta fos-b expression between sexually experienced

groups and sexually naïve groups. However, since most studies only look at delta fos-b

expression in males, we have added to the current literature on female molecular mechanisms of

the reinforcement of sexual behavior that there is an increase of delta fos-b expression in

sexually experienced hamsters compared to sexually naive female hamsters.

We were unable to support our second hypothesis that male and female sexually

experienced hamsters would have different levels of delta fos-b expression in the NAc. This

could have happened for many reasons. It is possible that there is no difference in upregulation of delta fos-b between males and females. This is the most likely explanation for our results. Our hypothesis was not based on any prior research, but more on our predictions based on differences between male and female sex behavior. Although there is a difference in sexual behaviors displayed between males and females, this is not indication the mechanisms for reinforcing behaviors are any different. The difference in the display of sexual behavior is much more easily DELTA FOS-B MECHANISMS IN NUCLEUS ACCUMBENS AFTER SEXUAL EXPERIENCE Sidibe 34

explained by the sexually dimorphic nucleus of the medial preoptic area (SDN-mPOA).

Evidence that the mPOA is sexually dimorphic comes from the fact that it has 3-7 times more volume of neurons in male rodents compared to females, and that lesioning the mPOA in males disrupts sexual behaviors, while leaving the female sexual behavior relatively unchanged

(Arnold & Gorski, 1984). The control of male sexual behaviors is found to be exclusively controlled by the mPOA (De Jonge et al, 1989; Everitt, 1990). Lesions to the medial pre-optic area of male rodents eliminates any ability to perform any sexual behavior (Hull and Dominguez,

2007). In addition, lesions to the NAc led to an increased post-ejaculatory interval, but had no effect on the ability for the mouse to copulate (Hull & Dominguez, 2007). This means that the control of sexual behavior and the rewarding of sexual behavior (Bradley et al, 2005; Pitchers et al, 2010) are controlled by two completely different pathways. In light of the evidence, it is unlikely for sex differences in types of expressed sexual behaviors to be reflected upon changes in delta fos-b expression in the NAc. This is more likely to be reflected in the sex differences in the mPOA.

Another methodological reason could be in using Western Blot as a technique to measure the level of protein. Most studies that look at delta fos-b have used immunohistochemistry (IHC) to measure the level of protein. Although the western blot technique is more quantitative in measuring protein levels than immunohistochemistry (IHC), it is possible that a difference in techniques would lead to different results. In a comparison between immunohistochemistry and western blotting in an assay for the presence of an oncogene in breast cancer, IHC and western blot only had a concordance rate of 83.1 % (Dittadi et al, 1993), and 82% in another study

(Piffanelli et al, 1996). However, this seems unlikely, as Western blot is found to be very a DELTA FOS-B MECHANISMS IN NUCLEUS ACCUMBENS AFTER SEXUAL EXPERIENCE Sidibe 35

sensitive method for picking up traces of protein, with detection possible of only 10 picograms of

protein (Lee et al, 2000), depending on the antibody used. Overall, the lack of sufficient evidence

points to the idea that delta fos-b is simply not regulated differently between males in females in

sexual reinforcement.

Implications of results -

We added to the literature that suggests that both males and females use delta fos-b to

reinforce sexual behavior. This means that targeting delta fos-b in NAc neurons could allow us to

manipulate the reward pathway in both males and females. The reward pathway, as discussed in

the introduction, can reinforce many types of rewarding behavior, including sugar/food intake

(Wallace et al., 2008), and drug use (Bozarth 1995; Nestler et al, 2001; Ito et al, 2003), in

addition to sexual behavior. In fact, hamsters and rats that overexpress delta fos-b in the NAc

show increased sexual reward (Hedges et al, 2009), and an enhanced response to cocaine use

respectively (Grueter et al, 2013). This indicates that manipulating delta fos-b levels may allow

us to target negative behaviors that result in too much reinforcement of a behavior. One such

negative behavior is pathological gambling. Pathological gambling has been shown to be

reinforced by similar mechanisms that are used in drug addictions, and has been shown to recruit

the mesolimbic pathway system during gambling tasks (Potenza, 2008). Interestingly, gambling

leads to a tolerance-like phenomenon of the mesolimbic pathway that is seen in drug addiction

(Reuter et al, 2005). Treatments that alter delta fos-b levels could be used to reduce the level of reinforcement of gambling.

By being one of the first experiments (to our knowledge) to test for the differences

between male and female mechanisms of sexual reinforcement, this research could pave the way DELTA FOS-B MECHANISMS IN NUCLEUS ACCUMBENS AFTER SEXUAL EXPERIENCE Sidibe 36 for future research attempting to elucidate the different mechanisms of sexual behavior reinforcement, and addiction in general. Many treatment methods currently use the same treatments for both males and females. If there are mechanisms of addiction (drug, sex, or otherwise) that differ between males and females, these drugs could be having differing levels of efficacy. Evidence shows that responses to different drugs can be different between male and female rats. Male rats that were in withdrawal from d-amphetamine administration decreased performance in object placement memory tasks and total locomotion compared to female rats, who only showed decreases in the object recognition performance task (Bisagno, Ferguson, &

Luine, 2003). These rats also displayed sexually dimorphic changes in neurochemistry, highlighted by a higher increase in norepinephrine in the NAc of males than in females. Sexual dimorphisms have also been observed in response to morphine and Mk-801 (NMDA receptor antagonist), where morphine induced greater behavioral responses in males, and MK-801 induced greater behavioral responses in females (D’Souza, Harlan, & Garcia, 2002). In addition, morphine caused a greater increase in c-fos expression in the striatum of males than in females.

Finally, estrogen, which is produced in higher amounts in female rats, has been found to enhances dopamine release from the NAc and dorsal striatum in response to amphetamine

(Becker, 1999). Since dopamine plays a large role in the reward and reinforcement of sexual behavior, this would suggest sexual differences in how rats respond to rewarding stimuli.

Whether this difference in mechanisms translates to other primates such as humans remains to be seen. The elucidation of mechanisms could lead to the development of drugs that take into account the sex differences of sexual behavior reinforcement.

DELTA FOS-B MECHANISMS IN NUCLEUS ACCUMBENS AFTER SEXUAL EXPERIENCE Sidibe 37

Limitations and Future Directions -

Although we only looked at delta fos-b as a molecular marker of sexual experience

dependent growth of the NAc due to time constraints, there are many more potential markers of reinforcement. Other proteins that we were considering were arc and brain-derived neurotrophic

factor (BDNF) There are many reasons to look at other proteins that are involved in the

reinforcement of rewarding behaviors. BDNF, a protein involved in synaptic and dendritic

growth, can increase the propensity for rewarding effects of drug abuse, and can mediate the

molecular changes induced by the drug (Nestler, 2002). Long-term potentiation through dendritic

spine growth after chronic cocaine use in rats has been shown to be mediated by BDNF

expression in the NAc (Nestler, 2002; Bolanos & Nestler, 2004). In addition, administering

cocaine to BDNF knock-down mice led to a decrease in the rewarding and locomotor effects of

the drug compared to normal controls (Hall, Drgonova, Goeb, & Uhl, 2003). This evidence

points towards a role of BDNF in the rewarding effects of these drugs, and its potential role in

the reinforcement of these behaviors.

Despite research indicating BDNF’s role in mediating reinforcement in drug addiction,

little research has been performed regarding its role in reinforcing sexual behavior. Exploring the

role of BDNF could be an interesting avenue for future research. In addition to BDNF, many

other molecular pathways that play a role in synaptic activity, second messenger systems,

neurotransmission, and receptor activity could be players in reinforcing sexual experience

(Bradley et al, 2005). The arc protein has received considerable attention for its role in the long-

term potentiation and memory consolidation. Inhibiting the activity of the arc protein in the

hippocampus using antisense oligonucleotides, which block the expression of proteins, leads to DELTA FOS-B MECHANISMS IN NUCLEUS ACCUMBENS AFTER SEXUAL EXPERIENCE Sidibe 38

the inability to maintain LTP, and impairs the consolidation of long-term memory in rats

(Guzowski et al, 2000). Exploring these molecular mechanisms could lend further clues to the similarities and differences to how sexual experience is reinforced between the sexes. In addition, future studies that scope for all the different types of mechanisms involved in the reinforcement of sexual behavior would help to generalize our results to human

In addition to studying other molecular markers of sexual reinforcement, future studies

could be directed at attempting to treat disorders of an excess or lack of expression of naturally rewarded behaviors. In humans, behaviors such as gambling (Reuter et al, 2005), sex, and sugar intake (Malik, Schulze, & Hu, 2006) can become problematic in high quantities. Looking at how the brain processes natural rewards in animal models could hint at treating these behaviors by

targeting the mesolimbic pathway. Rats who are given sugar water and sugar “chow” ad libitum

show increased evidence of withdrawal symptoms physiologically and behaviorally (Hoebel,

Avena, Bocarsly, Rada, 2009). This includes downregulated enkephalin expression due to

increased opioid receptor binding (presumably a response to increased opioid release from sugar

intake), and increased motivation to attain the stimulus even 2 weeks after abstinence. Other

signs of sugar’s potential role in addiction include the increase of dopamine in the NAc (Rada,

Avena, & Hoebel, 2005; Avena, Rada, Moise, & Hoebel, 2006), and increased opioid

dependence due to opioid receptor sensitization (Colantuoni, Rada, & McCarthy et al, 2002).

Since sugar intake is a naturally rewarded behavior in addition to sexual behavior, perhaps one could model sexual addiction after some of the physiological aspects of sugar addiction, such as increased activation of the mesolimbic pathway, and increased opioid dependence after excessive use. Research has already shown that targeting dopamine receptors with quinpirole (Cooper & DELTA FOS-B MECHANISMS IN NUCLEUS ACCUMBENS AFTER SEXUAL EXPERIENCE Sidibe 39

Al-Naser, 2006), and opioid antagonist naloxone (Rockwood & Reid, 1982) could reduce the preference for sugar water or food over normal (non-sugar) sustenance. Although there is no indication in the research that sugar intake reinforcement follows a similar pathway, future research could find out whether this is the case. Drugs that target the reward pathway by blocking dopamine or opioid receptors could be start off points for reducing the level of sexual activity in rodent models. Overall, developing sexual addiction models from another natural reward addiction model such as sugar intake could provide an avenue for translatable research that treats sexual addiction.

Finally, future studies could focus on making our research more generalizable. Two large issues in generalizing results from animal models to humans is the difference in how sexual behavior is represented in the brain, and the differences in environment. Although no comparative genomics study has compared hamsters to humans, rat and human genomes are found to have 80% identical genes, meaning that 80% of genes that codes for one protein in rats will code for the identical protein in humans (Emes, Goodstadt, Winter, & Ponting, 2003).

However, only three percent of our genome actually codes for protein (). The rest of our genome contains sequences that regulate the translation process of many different proteins, make pre- and post-translational modifications of proteins, among many other processes (). The differences of these processes may account for the large variation from species to species. Future studies could attempt to bridge the species gap by looking at the presence and expression of genes that are important in the reinforcement of sexual behavior. Unfortunately, studies on the genetic component of addiction only focus on genes implicated in drug addiction. Not surprisingly, genes that code for cholinergic receptors and products (Li & Burmeister, 2009) and DELTA FOS-B MECHANISMS IN NUCLEUS ACCUMBENS AFTER SEXUAL EXPERIENCE Sidibe 40 dopaminergic receptors and products (Foll, Gallo, Strat, Lu, & Gorwood, 2009) have been implicated in nicotine and other types of drugs respectively. Therefore, future studies must also work towards elucidating the genetic mechanisms of reinforcing sexual behavior. This could start with delta fos-b, but could also focus on the genes implicated in sexual behavior in Bradley et al (2005).

Generalizing our study must also take into account the difference in environment between hamsters and humans. One area of research where this is already occurring is in conditioned place preference. Rodent models of conditioned place preference suggest that rodents prefer sexual intercourse in places that they are used to having sex (Pfaus et al, 1990; Bell et al, 2010;

Been et al, 2013). This is interpreted as feeling a larger reward from places in which they have had sex. However, whether this conditioned place preference occurs in humans remains to be seen. In order for our study to be more generalizable, conditioned-place preference would need to be confirmed as a shared environmental reward enhancer. In addition, the motivation for human sexual intercourse varies widely from rodent motivations. Community standards, social pressures, cultural values, and socio-economic status have all been implicated in the motivation for sexual intercourse in humans (Brewster, Billy, & Grady, 1993). These are motivations that unfortunately cannot be replicated in a lab. Therefore, in order to make the results more generalizable, future research could look at higher level primates, such as monkeys. Although sex still occurs for mainly reproductive reasons in non-human primates (Seward, 1946), there is reason to believe that social motivations for sexual behavior are more present in non-human primates compared to lab rodents. Evidence suggests that primates can use sexual behavior as a form of social communication (Dixon, 2015). Unfortunately, primate sexual behavior does not DELTA FOS-B MECHANISMS IN NUCLEUS ACCUMBENS AFTER SEXUAL EXPERIENCE Sidibe 41 perfectly correspond to human motivations. However, our genetic similarity of around 95 percent

(Olson & Varki, 2003), and moderately overlapping social motivations, would provide a more generalizable model than rodent models for understanding the mechanisms for reinforcement of sexual behavior. However, ethical issues of using higher order primates would make it difficult to acquire enough subjects to examine the in depth mechanisms of sexual behavior reinforcement. For the time being, it may be more worthwhile to continue to develop more generalizable rodent models of sexual behavior by creating transgenic rodents and making the experiment as naturalistic as possible to mimic more natural conditions.

Conclusion:

Delta fos-b plays an important role in the reinforcement of sexual behavior. This reinforcement occurs through upregulation an of delta fos-b after multiple sexual experiences, which leads toan increase in dendritic spines and thus a more sensitized synapse. Targeting other natural behaviors such as gambling and food intake could be controlled by modulating the levels of delta fos-b in the NAc offers promising new lines of research. In addition to delta fos-b, other mechanisms of reinforcing sexual behavior (such as BDNF and the arc protein) remain to be investigated as possible differences between males and females. Investigating these other molecular mechanisms of sexual reinforcement has big implications, as it could provide a portal into treating disorders of natural behaviors that arise from excessive reward stimulation and reinforcement (such as excessive or deficient food intake, or sexual addictions). Future studies can borrow ideas from animal models of other natural behaviors (such as food eating) to look DELTA FOS-B MECHANISMS IN NUCLEUS ACCUMBENS AFTER SEXUAL EXPERIENCE Sidibe 42 into the effect of modulating proteins on behavioral measures. Translational research could then allow us to use these medications in humans.

DELTA FOS-B MECHANISMS IN NUCLEUS ACCUMBENS AFTER SEXUAL EXPERIENCE Sidibe 43

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Figure 1: Differences in delta fos-b expression in the NAc and control brain area (CP)

between naive

and

experienced 1A hamsters

1B **

1C 1D

Figure 1: Differences in delta fos-b expression in the NAc and control brain area (CP) between naive and experienced hamsters. Both male (F(1,2) = 25.20, p < .05) (A) and female (F(1,10) = 21.43, p = .001) (B) experienced hamsters showed a significant increase in delta fos-b expression in the NAc compared to naïve hamsters. In the caudate putamen (C,D) there was no significant increase in delta fos-b expression in experienced and naïve hamsters in either males (F(1,6) = .975, p > .05) (C) or females (F(1,10) = 0.851, p > .05) (D). (p < .05 = *, p <.01 = **).

DELTA FOS-B MECHANISMS IN NUCLEUS ACCUMBENS AFTER SEXUAL EXPERIENCE Sidibe 51

Figure 2: Comparing male and female differences

in delta

2A2B

fos-b

expressi

on in

the

NAc.

2C 2D

DELTA FOS-B MECHANISMS IN NUCLEUS ACCUMBENS AFTER SEXUAL EXPERIENCE Sidibe 52

Figure 2: Comparing male and female differences in delta fos-b expression in the NAc. There was no significant difference between male and females in expression of delta fos-b in the sexually experienced group in either the NAc (F = 1.51, p > .05) (A) or in the CP ((F = 0.107, p > .05)(B). Naïve hamsters (C and D) showed no differences between male and female delta fos-b expression in either the NAc (F(1,6) = 2.706, p > .05) or CP (F(1,7) = 1.326, p > .05) as well. DELTA FOS-B MECHANISMS IN NUCLEUS ACCUMBENS AFTER SEXUAL EXPERIENCE Sidibe 53

Figure 3: No effect of increased efficiency in female lordosis duration or latency throughout sexual experiences

3A 3B

Figure 3: No effect of increased efficiency in female lordosis duration or latency throughout sexual experiences. Lordosis duration (F(5,5) = 1.70, p > .05) (A) showed no increase over time, lordosis latency (F(5,5) = 2.17, p > .05) (B) showed no decrease over time over the 6 weeks of sexual experience. The lack of increase in lordosis duration, and the lack of decrease in lordosis latency suggests a lack of development of sexual efficiency over time (A,B)

DELTA FOS-B MECHANISMS IN NUCLEUS ACCUMBENS AFTER SEXUAL EXPERIENCE Sidibe 54

Figure 4: No effect in sexual efficiency found over time across mounts, intromission, and ejaculations.

4A 4B

4C

Figure 4: No increase in Sexual efficiency found after more sexual experiences. The number of mounts (F(5,5) = 0.10, p > .05) (A) and intromissions (F(5,5) = 0.32, p > .05) (B) showed no decrease in number over the 6 weeks of sexual experience. In addition, ejaculations (F(5,5) = 2.01, p > .05) (C) showed no increase over six weeks. The lack of increase in ejaculations, and decrease in mounts and intromissions shows a lack of increase in overall sexual efficiency (A-C).