ABSTRACTTo study the effects of oxytocin (OT), the use of intranasal OT has been more prevalent in recent years, however, the rewarding or reinforcing properties of it are not completely understood.  Recent studies, including those from Yutaka K, Shigeru W. 2016, Ramos et al. (2015), and Kent K. et al., 2013, have provided significant results in supporting the involvement of OT in more complex social behavior and cognition in humans, therefore, OT has been deemed as a potential therapeutic treatment for neuropsychiatric disorders characterized by social impairments.  The proposed study aims to investigate the reinforcing effects of oxytocin in Peromyscus californicus, a monogamous species commonly used in studying behavioral endocrinology, pair-bonding, and territorial behaviors, utilizing the conditioned place preference (CPP) paradigm.  In the three-chambered CPP apparatus, intranasal administrations of OT were used to repeatedly condition the female mice to a side chamber, which is an unfamiliar/neutral environment.  The final test revealed that OT-induced CPPs in the side chamber are developed only when paired with a social stimulus, neither the control or the OT groups produced significant results.INTRODUCTIONOxytocin (OT) is a hypothalamic neuropeptide that is synthesized in the paraventricular nucleus (PVN) and supraoptic nucleus (SON) in the hypothalamus.  It has been involved in a number of mammalian reproductive and social behaviors, such as lactation and parturition (Kosaki, Watanabe, 2016).  In reference to lactation, the paraventricular nuclei and supraoptic nucleus in the hypothalamus are stimulated, which signals to the posterior pituitary gland to produce the oxytocin.  During parturition, there is an increase in the concentration of oxytocin in the cerebrospinal fluid which facilities the birthing process and the establishment of maternal behavior.  In recent years, it has been recognized that the nanopeptide OT also serves as a crucial function as a neurotransmitter (a messenger released from a neuron at an anatomically specialized junction which then diffuses across a narrow cleft to affect one or two postsynaptic neurons, a muscle cell, or another effector cell) and/or neuromodulator (a messenger released from a neuron in the central nervous system, or in the periphery, that affects groups of neurons, or effector cells that have the appropriate receptors) within the central nervous system (Liberzon et al., 1997).  Evidence has asserted that the neuropeptide OT plays a critical role in social behaviors, which uncovers a potential treatment for social behavior deficits/neuropsychiatric disorders in humans including social anxiety disorder, schizophrenia, and autism spectrum disorder (Hung et al., 2017).  Seeing that these stress-induced psychiatric disorders commonly exhibit deficits in social processes and exaggerated responses to threats, there is an eager interest in investigating OT as a potential therapeutic agent.  The main reason for such interest being that OT possesses a variety of effects on social behaviors and behavioral responses to threat; these effects have been found to facilitate aspects of social bonding and cognition and exert anxiolytic effects (Steinman, 2016).Behavioral studies demonstrate that oxytocin plays an important role in regulating reproductive behaviors such as parturition, lactation,  parental care, and partner formation (OT provides a neuroendocrine template for the effects of multiple hormones on parenting) (Yutaka, 2016).  Other behaviors that are modulated by oxytocin include affiliation, ingestion, neuroadaptation, stress regulation, and memory, all of which are associated with the activation of limbic and paralimbic systems.  Although it is plausible for oxytocin to regulate the above behaviors by distinct neurobiological mechanisms, it is also rational to hypothesize that oxytocin may be able produce different behavioral effects through a more general mechanism, such as the reinforcing effects on the rewarding system.  Previous studies have exhibited the involvement of OT in modification of the reinforcing effects of abused recreational drugs such as opiates, cocaine, and methamphetamine (Baracz SJ, 2016).  On the contrary, recent studies on pair bonding in prairie voles have revealed OTs role in the enhancement of the formation of partner preference through its interactions with the dopaminergic projection in nucleus accumbens (Chappell AR, 2016).  Additionally, it has been observed that OT mediates the reinforcing and prosocial effect of ecstasy, a drug known to have prosocial and empathogenic effects (Yutaka, 2016).  These findings have sparked interest in the idea that social attachment and preference could possibly be understood in the same framework of neural circuits homologous to the circuits that are responsible in the formation of addiction to recreational drugs.Despite previous studies suggesting involvement of OT in the modification of reinforcing effects of abused drugs, it has yet to be confirmed if externally applied OT has reinforcing effects.  In the study by Ramos et al. (2015), it was found that OT can produce CPP if a conspecific animal was concurrently present during the conditioning sessions in which the place cue was paired with OT.  These findings suggest that OT catalyzes a social reinforcing property of conspecific animal for place cues. However, contrastingly, in the study by Kent et al. (2013) discovered that icv infusion of OT can produce CPP in female mice.  In this experiment, a conditioned social preference (CSP) paradigm was used resulting in the findings that pairing the presence of a same-sex conspecific with icv infusions of OT produced a formation of a conditioned preferences for that conspecific over another conspecific associated with vehicle infusions.  Whereas these recent studies suggest that OT has an intrinsic reinforcing property under specific circumstances, there has yet to be a systematic study that has assessed the reinforcing property of externally administered OT for both social and nonsocial conditioned stimuli, making it unsuitable to for accurate conclusions to be declared.With these factors in mind, the proposed study aims to investigate if externally applying OT has a reinforcing effect for social and nonsocial cues in female California mice.  A conditioned place preference (CPP) paradigm, a well-established behavioral method for assessing stimulus reward, was used for the experiment.  The OT was administered intranasally as it has a more direct relevance to clinical applications in humans as well as it being less stressful to the animals versus other invasive forms of injections (Bales et all., 2013).  The general CPP procedure consists of three phases: habituation, conditioning, and postconditioning.  The purpose of the habituation session is to familiarize the animals with nonspecific, possibly stressful aspects of the procedure such as handling, transport to the test room, and the novelty of exposing to apparatus in order to reduce the likelihood that such events will interfere with later learning about the cue-drug relationship.  Another purpose of habituation session is to assess the animal’s initial preference for the conditioned stimulus (CS) (e.g. side preference).  During the conditioning phase, animals are given one or more exposure to each of the stimulus alternatives (‘place’), preferably in a counterbalanced order.  Based on the design (unbiased or biased), the initially non-preferred place or a randomly selected place is consistently paired with the rewarding drug (e.g. intranasal injection of oxytocin), while the other place is consistently paired with vehicle (e.g. saline also through intranasal injection). The preference test is conducted twenty-four hours or later after the last conditioning trial.  This test consists of offering the animal a choice between the ‘places’ that contain the drug-paired and vehicle-paired CSs.  If the intranasal-injected oxytocin has rewarding effects, we would predict that the animals would have an increased amount of time in the drug-paired place and no such effects in the saline control group.METHODSSubjectsA total of 23 female Peromyscus californicus served as subjects from the breeding colony in the Department of Psychology of the University of Wisconsin, Madison.  The mice were maintained following the National Institute of Health Guide for the Care and Use of Laboratory Animals.  They were all experimentally naive before each experiment.  The animals were group-housed (2–3 per cage; 48 × 27 × 16 cm) with water and food available ad libitum throughout the experiments.ApparatusSix identical sets of three-compartment conditioned place preference (CPP) apparatuses (large polycarbonate testing cages (91 cm long × 46 cm wide × 43 cm high) divided into three equal chambers) were utilized for conditioning.  The two two side chambers of each apparatus were connected to the middle chamber by manually controlled, guillotine doors.  One side of the apparatus has black and white lines that run horizontally across the wall while the other side has black and white lines that run vertically up and down the other wall.  The different patterned stripes served as visuotactile stimuli that cues to differentiate the compartments in the CPP experiments.  All of the apparatuses had two transparent plastic lids that each covered half of the top of apparatus.  Circular white lamps was attached to a board directly behind the apparatuses above the center chamber to provide illumination. Both of the side compartments had mesh wire near the back of the apparatus, acting as a window separating the side compartments with a much smaller back compartment that is used to allow interaction between the subject and the stimulus mice.Stimulus miceStimulus mice used in each experiment were the same as the subject mice, female Peromyscus californicus.  They were sourced from the same supplier, but were unfamiliar to the subject mice.  The stimulus mice were matched to the subjects in each experiment with a 2:1 ratio, therefore, there were two stimulus mice used for two different subject mice, each in two different apparatuses.  The purpose of the stimulus mice is to see if the presence of a stimulus during the conditioning sessions will enhance the association between OT’s rewarding properties and the location.  In addition, they are used is to see determine if OT can produce a conditioned social preference.Drug 0.8 IU/kg of synthetic OT was administered intranasally for each subject.  The intranasal OT administration was performed by manually holding the animal in a supine position with one hand while the other dabbed drops of OT in and around the nasal cavity (alternating nostrils), using a Hamilton Gastight Syringe with a cannula attached to the tip of the needle.  ProcedureThe entire CPP procedure was conducted over a time period of eight days. Habituation – Baseline choice testThe pretest took place on day one, commencing with the placement of each subject mouse in the middle compartment of each apparatus.  After each mouse was given three minutes to acclimate to the novel environment, the two guillotine doors were manually raised and removed from the apparatus, allowing for the exploration of the two side compartments for 30 minutes.  Upon termination of each session, an experimenter immediately removed the subject mouse from the chamber and returned it back to its original holding cage.  The mean times spent in the side compartments were used as a baseline, preconditioning social preference (measured separately for each side using a stopwatch application on a windows computer).Conditioning Over the next 6 days (days 2-7), the subjects received social preference conditioning with intranasal OT as an unconditioned stimulus (US) and _____ as the the conditioned stimulus (CS).   Immediately after the intranasal OT was administered, each animal was placed in the side that was assigned (left or right) for the particular day based on its initial side preference in the pretest in its designated apparatus for 30 minutes.  When the 30 minutes was up, each animal was immediately taken out and returned it back to its original holding cage.Postconditioning On the the day following the final conditioning session (day 8), a choice test was conducted in a similar manner to the pretest.  Again, each mouse was given three minutes to acclimate to the novel environment and then the two guillotine doors were manually raised and removed from the apparatus, allowing for the exploration of the two side compartments for 30 minutes.  However, after the 30 minutes was over, the stimulus mice were then placed in the behind the mesh wire in the back of chamber (one on each side) for 10 minutes.The videos of the post conditioning will later be scored and the time in each ‘place’ (left or right chamber) will be recorded.  The data will then be imported into an Excel spreadsheet and a paired T-test will be ran to then determine if the effects of oxytocin significantly affects the time each subject spent in each of the ‘places’ (chambers).Additional tests Starting 2 days following the the posttest, the pretest followed by the same cycle of conditioning (6 days), and the posttest, were then repeated using different subjects each trial for a total of ___ experiments.RESULTSA two-way ANOVA was used to analyze the interaction between treatment (OT and saline) and stimulus (presence of stimulus mice during conditioning or no stimulus mice during conditioning).  There was not a significant did not reveal a significant interaction (F(1,19)=3.56, p > 0.01) between treatment (OT and saline) and stimulus (presence of stimulus mice during conditioning or no stimulus mice during conditioning).  Female California mice receiving saline injections did not form a CPP.  The mice also did not show a formation of a CPP for the side chamber associated with the OT injections either (figure ).  However, female mice did show a CPP for the side chamber associated with the OT drops when paired with a social stimulus (stimulus mice) (figure ).


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