Thesis

Electrically Evoked Dopamine Release in Virgin and Postpartum Rats:

Lack of Modulation by Nasal Oxytocin

Kyla Mace

The University of North Carolina at Chapel Hill

Spring 2016

A thesis presented to the faculty of The University of North Carolina at Chapel Hill in partial

fulfillment of the requirements for the Bachelor of Arts degree with Honors in Psychology.

Donita Robinson, PhD (Advisor)

Josephine Johns, PhD

Acknowledgments

I would like to thank my advisor Dr. Donita Robinson and co-advisor Dr. Josephine

Johns for their continuous support, encouragement, and advice. I would also like to thank Dr.

Tatiana Shnitko for her expert training in voltammetry and surgery and her patience in answering

my countless questions and concerns. Our lab manager Kaitlin Sullivan was of great help to me,

coming in many early mornings to help with rat breeding and cocaine injections. All of my

labmates deserve credit for providing moral support throughout the year. Finally, I am always

grateful to my parents for their unending support and love. This research was supported in part

Abstract

The oxytocin (OT) and dopamine (DA) systems are highly intertwined and have been implicated

in a number of similar behaviors, particularly social, sexual, and maternal behaviors. However,

the direct pharmacological effect of OT administration on the DA system is yet to be determined.

Additionally, although nasal OT is the primary route of administration in human research, it is

rarely used in animal studies, with researchers often relying on other routes for systemic OT

administration (e.g., intraperitoneal injection). The present study aims to fill this gap in the

literature by examining the effect of nasal OT on the DA system using fast-scan cyclic

voltammetry. Specifically, stimulated DA release in the nucleus accumbens was measured before

and after nasal OT administration in female rats. Because the peripartum period is rife with

neuroendocrine changes, both virgin and postpartum animals were used to examine how

pregnancy affects OT-DA interactions. However, no effect of OT was found, which is

inconsistent with past research and suggests an indirect mechanism by which OT affects DA and

DA-driven behaviors. Parity status was found to have a significant effect on baseline levels of

evoked DA release, with postpartum animals showing larger concentrations of DA compared to

virgin females. This corroborates past studies finding alterations in the DAergic system

Electrically Evoked Dopamine Release in Virgin and Postpartum Rats:

Lack of Modulation by Nasal Oxytocin

Our drive to form social bonds between friends, family, and romantic partners relies on a

complex neural circuitry, partially involving the oxytocin (OT) and dopamine (DA) systems.

These two neurochemicals are highly involved in motivational behavior, and their interaction has

been implicated in social-cognitive processing. However, the details of this interaction are still

under investigation, and OT’s effect on motivational behavior (and the DAergic system by

extension) seems to vary depending on the context. Although DA appears to be involved in all

types of motivational cues, OT seems to enhance the salience of social cues in particular. It has

been hypothesized that the dysregulation of these two systems can lead to maladaptive behaviors,

such as drug abuse and child neglect, where naturally motivational social cues are devalued and

drug cues are overvalued. In this paper, I will first provide a brief overview of the OT and DA

systems, their circuitry, and the behaviors in which they are involved, followed by a description

of how the two systems are connected. Next, I will relate animal findings to human research and

provide a brief introduction to fast-scan cyclic voltammetry, the electrochemical technique I will

use to measure DA release. Finally, I will introduce my general plan for investigating the

interaction of the OT and DA systems.

Oxytocin and Dopamine: Circuitry and Interaction

OT exerts a wide range of effects, both behavioral and biological, likely due to its ability

to act as both a neurotransmitter in the nervous system and a hormone throughout the body. On a

physiological level, it plays a fundamental role in lactation, erection, ejaculation, and triggering

labor (Gimpl, Fahrenholz, & Gene, 2001). In the central nervous system, OT has been implicated

caregiving (for review, see Lee, Macbeth, Pagani, & Scott Young, 2009). This range of effects

may be due to OT’s widespread presence throughout the brain. OT is primarily synthesized in

the paraventricular nucleus (PVN) and supraoptic nucleus (SON) of the hypothalamus (Gimpl et

al., 2001). From there, the SON primarily projects to the pituitary to release OT into the

bloodstream, but the PVN has a wide range of projections, including the olfactory bulb, medial

preoptic area (MPOA), nucleus accumbens (NAc), amygdala, hippocampus, and ventral

tegmental area (VTA) (Rutherford, Williams, Moy, Mayes, & Johns, 2011).

OT has been linked with a number of neurochemical systems, including serotonin

(Marazziti et al., 2012), nitric oxide (da Silva, Cedraz-Mercez, & Varanda, 2014), and GABA

(Lonstein, Maguire, Meinlschmidt, & Neumann, 2014). Additionally, OT and DA have been

found to share some circuitry and are involved in similar behaviors. DA plays a fundamental role

in motivational behaviors, including reward learning (Day, Roitman, Wightman, & Carelli,

2007), drug administration (Di Chiara et al., 2004), social and sexual behavior (Brom, Both,

Laan, Everaerd, & Spinhoven, 2014; Insel, 2003), and maternal caregiving (Love, 2014). A

major source of DA is the VTA, which projects to a number of forebrain structures, including the

olfactory tubercle, prefrontal cortex, and NAc, the last of which being the main focus of the

present study. The NAc has been strongly implicated in motivational behavior, and the amount

of effort an animal will exert to achieve a reward is dependent on extracellular DA in the NAc

(for review, see Salamone, Correa, Farrar, & Mingote, 2007). In addition, NAc DA transients, or

burst firing of DA neurons, are thought to be important in cue-reward associations. Through the

course of Pavlovian conditioning, where an unconditioned stimulus (e.g., a light) is presented

before an unconditioned reward (e.g., a food pellet), DA transients shift from occurring after the

predictive value of the cue (Day et al., 2007). NAc DA also plays a fundamental role in maternal

behavior, especially appetitive behaviors (goal-directed behaviors), such as pup retrieval, which

is impaired after administration of a DA antagonist to the NAc (Keer & Stern, 1999). DA release

in the NAc has also been found during pup licking/grooming (Champagne et al., 2004) and in

response to pup cues (cries and odors) in hormonally primed virgin females (Afonso, Shams, Jin,

& Fleming, 2013).

The DAergic and OTergic systems are interrelated in a number of ways. The PVN

projects to both the VTA and the NAc, and the MPOA sends strong projections to the VTA (for

review, see Numan & Stolzenberg, 2009). Both the VTA and NAc express OT receptors, and

many OT cells in the MPOA, PVN, and SON express DA receptors in return, indicating a

reciprocal relationship between the two systems (Baskerville, Allard, Wayman, & Douglas,

2009; Vaccari, Lolait, & Ostrowski, 1998). OT receptors are not found on the DAergic cells in

the VTA, however, indicating that their relationship may be indirect (Yoshida et al., 2009). Even

so, their neurobiological interaction is plausible, given that both neurochemicals have been

implicated in similar behaviors, including social, sexual, and maternal behavior.

OT’s influence on the DA systems can be facilitatory or inhibitory, depending on the

context. For example, infusing DA into the subthalamic nucleus of the hypothalamus enhanced

conditioned place preference, but adding OT abolished this effect (Baracz & Cornish, 2013).

This suggests that OT in this area inhibits the motivating effect of DA in the conditioned place

preference task. Additionally, OT has been found to suppress some effects of drugs of abuse (for

review, see McGregor & Bowen, 2012). Peripheral OT administration has been shown to reduce

the locomotor effects of methamphetamine and cocaine, both of which increase DA release and

These effects may result from OT’s inhibitory effect on the DA system, given that OT reduced

methamphetamine-induced activity in the NAc (Carson et al., 2010) and inhibited the increased

DA utilization found after cocaine administration (Kovàcs et al., 1990).

However, in other contexts, OT appears to exert an excitatory effect on the DA system

and its related behaviors. Although adding OT diminishes a DA-induced conditioned place

preference, the same effect is not found in a conditioned partner preference task (Triana-Del Rio

et al., 2015). In this experiment, male rats were co-housed with a same sex partner and received

saline, a DA receptor agonist, or a combination of a DA receptor agonist and OT. When later

presented with either their same-sex partner or a sexually receptive female, both the DA and

DA/OT animals preferred their male partner, whereas saline animals preferred the female,

suggesting an alteration in either social or sexual behavior. In this case, adding OT did not

eliminate the partner preference like it did for conditioned place preference. In terms of maternal

behavior, administering OT to virgin female rats increases pup-directed behaviors, such as

licking, nursing, pup retrieval, and nest-building, all behaviors that rely on DA (Fahrbach, 1984).

Infusing an OT antagonist into the VTA of postpartum dams reduced DA release in the NAc

during pup interaction, suggesting that pup-induced DA release is dependent on OT (Shahrokh,

Zhang, Diorio, Gratton, & Meaney, 2010).

It seems as though OT’s influence on DA depends on the type of reward involved,

possibly due to parallel brain systems used for processing non-social and social rewards. Either

reward system can be strengthened by exciting the DAergic system, either by administering a

DA agonist or drugs of abuse that act on the DA system, like methamphetamine and cocaine.

However, the OTergic system appears to devalue non-social rewards and increase the salience of

drugs of abuse, OT appears to have an inhibitory effect on the DA system and the behaviors

involved. However, for social and/or sexual rewards, such as a partner preference and maternal

behavior, OT appears to exert an excitatory effect on the DA system. Although there is a wealth

of research investigating OT-DA interactions in awake, behaving animals, much less is known

about the basic pharmacological effect of exogenous OT on the DA system.

Oxytocin Administration in Animal and Human Studies

Animal studies tend to use infusion or injection to administer OT, but these techniques

are too invasive for human studies. Instead, intranasal OT has been used widely in the human

literature to modulate social functioning. Intranasal OT may increase the salience of socially

relevant cues, including positively and negatively charged faces (Groppe et al., 2013), infant

laughter (Riem et al., 2012), and both infant and sexual images (Gregory, Cheng, Rupp,

Sengelaub, & Heiman, 2015). Additionally, intranasal OT has been found to improve symptoms

of conditions that impair social functioning, such as schizophrenia (Feifel et al., 2010) and

autism (Domes et al., 2013; Guastella et al., 2010). Finally, as mentioned earlier, intranasal OT

has been proposed as a possible treatment for drug addiction, due to its ability to reduce the

salience of drug cues (for review, see McGregor & Bowen, 2012).

Because intranasal OT is so widely used in human studies, its use in animal studies would

allow for a more accurate translation of animal findings to human research. Additionally,

intranasal administration has some practical benefits that other administration routes lack.

Intranasal administration allows OT to enter the central nervous system through the olfactory

epithelium, largely bypassing the blood-brain barrier, which can prevent as much as 99.998% of

subcutaneously administered OT from reaching the central nervous system (Mens, Witter, & Van

of rats, where it has only a 2 minute half-life. However, in the cerebrospinal fluid, its half-life

can be extended to up to 19 minutes (Mens et al., 1983). For these reasons, intranasal OT may be

more effective in elevating central OT levels for extended periods of time.

Although it is not commonly used in animal studies, intranasal (in non-human primates)

and nasal (in rodents) OT appears to exert similar effects as other routes of administration. As

with injections and intracerebral infusions, nasal OT has been found to increase central and

peripheral OT levels in rats and mice, although the exact route by which this occurs is unclear

(Neumann, Maloumby, Beiderbeck, Lukas, & Landgraf, 2013). Research on the behavioral

effects of intranasal OT in animals appears to mirror findings from the human literature.

Intranasally applied OT reduced the hypothalamic-pituitary-adrenal axis stress response to social

isolation in squirrel monkeys (Parker, Buckmaster, Schatzberg, & Lyons, 2005) and facilitated

social bonding in marmoset monkeys (Smith, Ågmo, Birnie, & French, 2010). However, little

research has been performed investigating how intranasal or nasal OT affects the DA systems in

animals.

Fast Scan Cyclic Voltammetry

The present study examines how nasal OT affects NAc DA release, using fast-scan cyclic

voltammetry (FSCV). FSCV allows for a higher spatial and temporal resolution than other

techniques such as microdialysis. This electrochemical technique involves applying a range of

electrical potentials to an electrode placed in the brain region of interest; as the chemicals around

the electrode undergo oxidation-reduction reactions at the various potentials, the changes in

current are measured to deduce the concentration of specific chemicals (Robinson, Venton,

Heien, & Wightman, 2003; Robinson & Wightman, 2007). For DA, the waveform of the applied

at 10 Hz throughout a recording session. The applied potential creates a large background

current, which can be subtracted from the recorded voltammograms to reveal smaller changes in

current. Because compounds have specific potentials at which they oxidize or reduce, the

background-subtracted voltammogram can be identified as a specific molecule, allowing for

measurement of the chemical of interest. Specifically, DA oxidizes to its ortho-quinone at

approximately 0.6 V and the ortho-quinone reduces back to DA at approximately -0.2 V

(Robinson & Wightman, 2007). This exact pattern is not shared with other compounds such as

serotonin or nitric oxide. However, norepinephrine oxidizes and reduces at the same potentials as

DA, making it important to consider the anatomical position of the electrode when identifying

norepinephrine and DA using voltammetry (Robinson & Wightman, 2007).

FSCV can be used in freely moving rats to measure real-time neurochemical changes as

the animal behaves and interacts with its environment, but it can also be used in anesthetized

animals to answer pharmacological questions. To measure DA release in the NAc in anesthetized

animals, a stimulating current is applied to the VTA and the resulting DA release is measured at

its terminals in the NAc. In this way, the neurobiological effects of a pharmacological agent

(such as OT) can be isolated from its behavioral components and environmental influences can

be eliminated.

Project Plan

To investigate OT’s interactions with the DA system, electrically stimulated DA release

in the NAc was measured before and after nasal OT administration (or its vehicle as a control).

Virgin females and postpartum dams were used to determine whether pregnancy alters OT’s

effect on NAc DA, given that the neuroendocrine system, OT included, undergoes a wide array

increases DA release in the NAc. Past studies have found that infusing OT into the VTA

increases DA release in the NAc of awake animals, in both male and female animals (Melis et

al., 2007; Shahrokh et al., 2010), but whether this occurs after nasal OT in anesthetized animals,

as well as postpartum dams, is yet to be established.

Methods Animals

Virgin female (250-430g) Sprague-Dawley rats (Charles River, Raleigh, NC) were pair

housed on a 12-hour light-dark schedule. Female rats to be bred were single housed on a reverse

12-hour light-dark cycle (lights off at 0900) for a 2-week habituation period, after which they

were housed with breeder males. Gestation day (GD) 0 was designated as the day a sperm plug

was found. Pregnant females were weighed daily throughout their gestation. On GD 1, females

were returned to single housing and kept under the reverse light-dark cycle until GD 7, when

they were moved to a room with regular light cycle (lights on at 0700) for the rest of the

experiment. This procedure generally causes dams to deliver during daytime hours on GD 21

(Mayer & Rosenblatt, 1998). On delivery day (postpartum day (PPD) 0), litters were culled to 4

males and 4 females. The final weights of postpartum animals ranged from 213 to 323g. All rats

received water and food ad libitum. All procedures were conducted under federal and

institutional animal care and use committee guidelines for humane treatment.

Surgery

Virgin females underwent surgery at least one week after arriving in the animal facility, and

postpartum dams underwent surgery on PPD 1. Litters from dams were euthanized 30 minutes

prior to the dams’ anesthesia administration to remain consistent with past studies from the Johns

conducted. Rats were anesthetized (1.5g/kg urethane, i.p.) and placed in a stereotaxic frame.

Electrodes were implanted as previously described (Shnitko & Robinson, 2014); electrode

placements (from bregma) were -5.0mm and ML -1.2mm for the stimulating electrode and AP

+1.7mm and ML +1.8mm for the carbon fiber electrode. The stimulating electrode (bipolar,

parallel, stainless steel, 0.2 mm diameter/tip; Plastics One, Roanoke, VA) was initially placed in

the VTA at DV -8.0mm, and the carbon fiber electrode (length 60-115μm) was initially placed in

the NAc core DV -6.0mm. Both electrodes were lowered to optimize DA signal, with final

placements ranging from -8.1mm to -9.0mm for the stimulating electrode and -6.0mm to -6.3mm

for the carbon fiber electrode. A reference electrode (Ag/AgCl) was placed in the left hemisphere

and secured with a stainless-steel screw and dental cement.

Voltammetry and Experimental Procedure

The applied potential was ramped from -0.4 V to +1.3 V and back to -0.4 V every 100

ms. A stimulating current was applied to the VTA (24 biphasic square-wave pulses, 2 ms per

phase, 260.3 – 304 μA, 60 Hz); the stimulation current was adjusted to optimize the DA signal

and varied from animal to animal. Three evoked signals, collected 5 minutes apart, were

measured before OT or vehicle administration to determine the animal’s baseline level of

stimulated DA release. Following nasal treatment (10 μL per nostril of 1 mg/ml OT or its

vehicle), 12 evoked signals were collected five minutes apart, totaling an hour of voltammetric

recording. This time frame was chosen because brain OT levels peak at approximately 30 – 60

minutes after either intraperitoneal or nasal OT administration (Neumann et al., 2013). In order

to convert current to DA concentration, electrodes were calibrated in vitro post-experiment, as

previously described (Robinson & Howard, 2009). In short, current was recorded from each

concentration of DA (1μM). Electrodes from 10 animals were preserved for calibration, and

calibration factors (μM/nA) for the remaining electrodes were estimated from these calculations.

Drug Preparation

1mg/mL OT solution was prepared by dissolving synthetic OT (Bachem Americas, Inc.,

Torrance, CA) in a saline and 0.1% acetic acid solution, as OT requires a mildly acidic

environment in order to dissolve. Aliquots were kept frozen at -80°C until the time of the

experiment.

Analysis

Voltammetric analysis. Before analysis, data files were renamed in order to mask the time point at which the file was taken (i.e., before or after drug administration) to reduce researcher

bias during analysis. Voltammetric data was analyzed as previously described (Shnitko &

Robinson, 2014). DA was identified in the voltammetric current using color plots and cyclic

voltammograms. An average background voltammogram was taken from the 5 seconds

preceding stimulation and was subtracted from voltammograms taken during and after

stimulation to create background-subtracted voltammograms. No 2D smoothing was applied to

the data. Current-versus-time traces were used to determine peak current (nA) for each

stimulation. Calibration factors collected post-experiment were used to convert peak current to

peak concentration (μM), which will be referred to as [DA]max.

Dopamine signal modeling. To investigate whether DA release and uptake differed between virgin and postpartum groups, DA release and clearance in baseline recordings were

examined as previously described (Shnitko, Spear, & Robinson, 2015) using the

Michaelis-Menten kinetic equation (Wu, Reith, Wightman, Kawagoe, & Garris, 2001). This equation

pulse of stimulation ([DA]p, nM), the frequency of electrical stimulation (f, Hz), the velocity of

DA transporters (Vmax, nM/sec), and the affinity of those transporters (Km, nM). For each

baseline recording, a model of the current-versus-time plot was generated using this equation. Km

was held at 200nM and [DA]p and Vmax were modified until the model resembled the

experimental data, with a correlation coefficient of at least 0.9. For each animal, average [DA]p

and Vmax measurements were calculated from the three baseline files and were then compared

between virgin and postpartum groups, collapsing across nasal treatment groups.

Statistical analysis. To examine maximum evoked DA concentration over the course of the experiment, each rat’s 15 data points were condensed to five time points by averaging [DA]max

from three consecutive files at a time, leaving each rat with five total measurements: one baseline

measurement and four post-administration measurements. To examine how postpartum status

affects the concentration of DA release, concentration of DA released per pulse of stimulation,

and the velocity of DA transporters, three Mann-Whitney Rank Sum tests were performed (due

to non-equal variance between groups), comparing [DA]max, [DA]p, and Vmax between virgin and

postpartum groups, collapsing across nasal treatment groups.

To examine changes in DA signal following OT treatment, the data for each rat was

normalized to baseline by calculating the percent change from baseline for each of the four

post-administration measurements. A two-way repeated measures ANOVA was performed with

percent change from baseline as the dependent variable, group as a between-subjects factor

(OT/virgin, vehicle/virgin, OT/postpartum, vehicle/postpartum), and time as within-subjects

Results Baseline Dopamine

To determine how parity affects evoked DA release, we examined [DA]max at baseline in

virgin animals (n=6) and postpartum dams (n=7), collapsing across nasal treatment groups. Of

the postpartum dams, five were run the day after delivery (PPD 1), and two were run on PPD 3.

All animals included in this analysis received 2.24-2.26V stimulation (289-304μA); two animals

(both OT-treated virgin females) received 2.2V (260.3μA) stimulation and were removed from

this analysis. The baseline concentration of evoked DA release in postpartum dams (Mdn=0.941

μM) was over five times larger than virgin females (Mdn=0.123 μM), as shown in Figure 1. This

difference was statistically significant (Mann-Whitney U=2.0, p=0.005). As shown in Figure 2,

[DA]p also varied significantly between virgin and postpartum groups as well (Mann-Whitney

U=2.0, p=0.008), with postpartum dams releasing more DA per stimulation pulse (Mdn=62 nM)

than virgin animals (Mdn=8.833 nM). Also illustrated in Figure 2, although Vmax did not vary

significantly between parity groups, there was a trend toward significance (Mann-Whitney

U=7.0, p=0.051), with postpartum animals showing reduced velocity of DA transport

(Mdn=1600 nM/sec) compared to virgins (Mdn=826.667 nM/sec). Figure 3 shows color plots

from representative animals to illustrate these differences.

Change from Baseline

To examine the effect of nasal administration of OT or its vehicle on DA release, the

percent change from baseline was determined for each 15 minute averaged epoch. Regardless of

nasal treatment group or parity status, there was no change from baseline, as shown in Figure 4.

time [F(3, 30) = 0.587, p=0.628] on percent change from baseline, nor was there an interaction of

group and time [F(9, 30) = 0.361, p=0.945].

Discussion

Nasal Oxytocin Does Not Modulate Stimulated Phasic Dopamine Release

Although the interactive role of OT and DA in behavior is under thorough investigation,

the direct pharmacological effect of OT administration on the DA system is relatively unclear. In

the present study, the effect of nasal OT administration on stimulated DA release in the NAc was

examined. Although we predicted that OT would increase DA release, no effect was found. This

runs in contrast to the behavioral studies upon which this project was designed, which found that

OT exerted either a facilitatory or inhibitory effect on the DA system, depending on the

behavioral context under investigation.

We anticipated a simple, direct effect of nasal OT on DA release, but it appears that the

process may be more complex. Indeed, even the basic assumption that OT directly facilitates

social behavior has been questioned in the literature. Though OT is often defined as the “moral

molecule” or the “hug hormone,” its effects on behavior may be less than straightforward. Many

behavioral studies tend to consider only a top-down effect of OT on social behavior; i.e., changes

in behavior are due to OT’s direct effect on the central nervous system. However, OT’s effect on

peripheral organs is not to be dismissed, and bottom-up processes should be taken into

consideration. Beyond the central nervous system, OT receptors are present throughout the body,

including the heart and cardiovascular system, the kidneys, mammary tissues, and the

reproductive tract of both males and females, all of which could be affected by systemic OT

administration (Gimpl et al., 2001). Indeed, OT administration has been known to reduce body

Additionally, organs in the periphery may influence OT release in the nervous system, as both

the heart (van Lieshout, Wieling, Karemaker, & Eckberg, 1991) and the kidneys (Ciriello, 1998)

have afferent projections to the hypothalamus. Vaginocervical stimulation has been found to

increase OT release in the olfactory bulb (OB) and enhance social recognition memory

(Larrazolo-López et al., 2008), so it is entirely possible that systemic OT’s effect on organs can

indirectly influence central nervous system activity and behavior by extension.

In addition to OT’s peripheral effects, OT has been found to exert an anxiolytic effect

(Yoshida et al., 2009). Like its effects on social behavior, this reduction in anxiety may be due to

the complex interaction of its central and peripheral effects. It is also important to consider

whether OT’s social behavior effects may be partly due to its anxiolytic nature (for review, see

Churchland & Winkielman, 2012). As Churchland and Winkielman note, the basis of social

behavior is one’s willingness to approach another, which is sensitive to anxiety levels. In this

way, an increase in social behavior may be just one result of a general reduction in anxiety.

All of the above illustrate the complex nature of OT’s effects on social behavior. Using

nasal OT as our route of administration does little to simplify the problem, as nasal OT’s route

into the nervous system has been disputed. The potential benefits of nasal administration of

peptides were discussed earlier, but the exact route by which nasally administered neuropeptides

enter the brain has been disputed (for review, see Leng & Ludwig, 2015; Quintana, Alvares,

Hickie, & Guastella, 2015). The most likely route of access is the olfactory pathway, in which

molecules reach the OB via olfactory fibers, and then are transferred to brain tissue or

cerebrospinal fluid (Quintana et al., 2015). Through this pathway, it is theoretically possible that

nasal OT could reach more centralized brain regions, as the OB has projections to the amygdala

under what specific circumstances. A neuroimaging study found that nasal OT only activated the

primary olfactory system, some brain stem regions, and sensory afferents, suggesting that OT

was not able to penetrate into more centrally located brain regions (Ferris et al., 2015). In

contrast, a microdialysis study found that nasal OT increased OT levels in the amygdala and

hippocampus, suggesting that it is indeed reaching central brain regions (Neumann et al., 2013).

However, the implantation of microdialysis probes causes significant tissue damage, and plasma

OT may have been unintentionally measured as blood vessels were ruptured.

This raises the question of how nasal OT has been found to alter social behavior, if it may

not reach past the OB, brain stem, and sensory afferents. Again, it is important to note the

interplay of top-down and bottom-up processes. Even if nasal OT is unable to penetrate into

deeper brain regions, its superficial effects may be sufficient to elicit social behavior changes. Its

effects on the OB may alter how olfactory stimuli is processed, leading to altered social

behavior. Indeed, infusion of OT into the OB is sufficient to enhance social recognition (Dluzen,

Shang, & Landgraf, 2001).

Beyond the complexity of the OT system, a few factors of our study may have

contributed to the lack of effect. A significant constraint is our use of normal virgin and

postpartum animals; it is possible that we encountered a ceiling effect, whereby administering

OT to a normal OT system had little impact. One next step will be to look at animals with

disrupted OT systems. For example, postpartum dams treated with cocaine during gestation show

a number of OT disruptions, including lower levels of OT in various areas of the brain, including

the VTA (Johns, Lubin, Walker, Meter, & Mason, 1997), and reductions in OT receptor binding

(Jarrett, McMurray, Walker, & Johns, 2006). These changes may be partly responsible for the

Noonan, & Zimmerman, 1994). If we administer the same experimental procedure to

cocaine-exposed dams and see no effect, it will provide further evidence that nasal OT does not directly

affect the mesolimbic DA system. However, if an effect is found, it would suggest that a ceiling

effect was responsible for the results from the present study.

In addition to the possible ceiling effect of OT, the present study contained some

methodological limitations. First, we measured evoked DA release by electrically stimulating the

VTA. Although this provides the benefit of controlling the environment under which each

measurement is taken, it is in no way a realistic representation of normal brain activity, and the

relatively large current applied to the VTA may mask more subtle changes that could be

occurring. Secondly, we used one acute administration of nasal OT, which may not be sufficient

to elicit noticeable changes, and follow-up experiments may utilize repeated treatments before

the experiment.

Pregnancy Affects Stimulated Dopamine Release at Baseline

When comparing baseline measurements between animal groups, we found significantly

higher DA concentrations in early postpartum dams compared to virgin animals. This is a

plausible finding in consideration with other studies examining the role of the DA system in the

postpartum period. In order to prepare for the onslaught of novel pup-stimuli and the

energy-intensive task of caring for a litter, the peripartum period is rife with changes in the DA system.

However, the precise changes that occur in this time are still under investigation. Postpartum

animals have been found to exhibit reductions in 3,4-Dihydroxyphenylacetic acid (DOPAC), a

metabolite of DA, indicating alterations in DA turnover (Byrnes, Bridges, Scanlan, Babb, &

Byrnes, 2007; Olazábal, Abercrombie, Rosenblatt, & Morrell, 2004). A similar effect is found

Similarly, we found a trend toward a reduction in the velocity of DA transporters in postpartum

dams. Together, these findings suggest that DA is not cleared as readily from the synapse in

postpartum dams as it is in virgin animals. Additionally, we found that more DA was released

per pulse of electrical stimulation in postpartum dams. It is unclear why this may be, as little

research has been performed examining basal DA content in the NAc in the early postpartum

period. Tissue studies have found similar levels of DA content between virgin animals and

postpartum dams (Byrnes et al., 2007), but in vivo experiments have revealed lower basal DA

levels in postpartum dams (Afonso et al., 2009; Afonso, King, Novakov, Burton, & Fleming,

2011; Afonso et al., 2013). Our findings provide additional information as to how the

mesolimbic DA system changes in the early postpartum period, and follow-up experiments can

investigate the exact mechanisms at play.

Conclusion

Although we anticipated a direct excitatory effect of nasal OT on NAc DA release, this

was not the case, suggesting a more complex relationship between the two systems. However,

we did find an effect of postpartum status on basal DA release, contributing to the understanding

of changes in the mesolimbic DA system in the early postpartum period. Further experiments

using cocaine-treated dams will reveal whether nasal OT exerts a different effect on animals with

References

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Figures

Figure 1. Evoked DA at baseline between virgin and postpartum animals. Postpartum animals

released significantly higher concentrations of DA at baseline to the same electrical stimulation

compared to virgin animals (p=0.005). The second and third quartiles are represented by dark

and light gray, respectively. 0

0.2 0.4 0.6 0.8 1 1.2 1.4 1.6

Virgin (n=6) Postpartum (n=7)

[D

A

]max

, μ

M

Figure 2. DA released per pulse ([DA]p) and velocity of DA transporters (Vmax) derived from

electrically evoked DA signals in virgin and postpartum animals. VTA-NAc neurons in

postpartum animals released significantly more DA per pulse of electrical stimulation than virgin

animals (p=0.008). The velocity of DA transporters (Vmax) in postpartum animals was reduced

0 20 40 60 80 100 120

Virgin (n=6) Postpartum (n=7)

[D

A]

, nM

0 500 1000 1500 2000 2500 3000

Virgin (n=6) Postpartum (n=7)

V

ax

, nM/

se

c

compared to virgin animals. This difference, although not statistically significant, was a strong

Figure 3. Representative color plots (left) and current vs. time traces (right) from a virgin female

(top) and postpartum dam (bottom). Color plots display changes in current (color) at a range of

applied potentials (y-axis) over time (x-axis). Inset, DA cyclic voltammogram from respective

Figure 4. Evoked DA concentration as a percent of basal measurements in virgin and postpartum

rats after nasal treatment of either OT or vehicle. [DA]max was averaged into four 15-minute

epochs. No differences were observed between groups or over time. 50%

60% 70% 80% 90% 100% 110% 120%

0-15 15-30 30-45 45-60

Perc

en

t

C

h

an

ge

from

B

as

el

in

e

Time After Treatment (min)

OXT/Virgin (n=4)

OXT/Postpartum (n=4)

VEH/Virgin (n=4)