Oxytocin Antagonist Affects Prepulse Inhibition in Prairie Voles

Crystal Vardakis a, Julia Palmer a, Karen L. Bales a*

a Department of Psychology, One Shields Ave. Davis, CA 95616 USA

cavardakis@ucdavis.edu a, jfpalmer@ucdavis.edu b, klbales@ucdavis.edu c

* Corresponding author at: Department of Psychology, One Shields Ave. Davis, CA 95616 USA

Tel.: +1 530 754 5890

E-mail address: klbales@ucdavis.edu (K.L. Bales).

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Abstract

There is evidence that impaired oxytocin (OT) biology is involved in the pathophysiology of

schizophrenia, in which patients also show deficits in sensorimotor gating. Percent prepulse

inhibition (% PPI) is a measure of sensorimotor gating often used in rodent studies, and is shown

to be modulated by OT. The goal of this study was to perform an initial, exploratory

investigation into the relationship between OT and PPI in prairie voles, a socially monogamous

rodent frequently used as an animal model of psychopathologies which involve social deficits.

Intranasal administration of OT is currently being proposed as a treatment for these

psychopathologies, including schizophrenia and autism. In this study, voles were divided into

groups which showed higher (>60%) or lower (<60%) levels of % PPI. Voles which had low %

PPI were administered intranasal OT and saline, while voles which had high % PPI were

administered a centrally acting OT receptor antagonist (central OTA or "COTA"), a peripherally

acting oxytocin antagonist ("POTA"), and saline via intraperitoneal injection. Both OTAs

decreased baseline startle amplitude in males, but not in females. Intranasal OT, compared to

saline, did not significantly improve % PPI. In males, COTA, compared to saline, significantly

worsened % PPI while POTA did not. For females, POTA worsened % PPI while COTA did not.

These results suggest that OT administered intranasally at this dose does not improve % PPI.

These results do however, support the involvement of the OT system in PPI and suggest that

there are sex differences in the role of OT in the startle response and in pre-pulse inhibition.

Key words: oxytocin, schizophrenia, autism, pre-pulse inhibition, startle

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1. Introduction

Oxytocin (OT) is a neuropeptide associated with attachment, parenting, and social

cognition in both humans and animals. The prairie vole (Microtus ochrogaster) forms pair-

bonds, exhibits biparental care, and also demonstrates a different distribution of OT and

vasopressin (AVP) V1a receptors in the brain than closely related polygynous species [1-5]. The

socioemotional bonding displayed by the prairie vole makes this species an excellent tool for

studying the neurobiological underpinnings of both healthy and disrupted social processing [6,

7]. However, very little data are available on sensorimotor gating in this species [8].

There is evidence that disruptions in OT biology may contribute to the pathophysiology

of schizophrenia [9-11] and autism [12-15]. The role of endogenous OT in

schizophrenia, however, is not well understood. For instance, in both male

and female patients there appears to be an association between peripheral

OT and symptom severity. Rubin and colleagues discovered that although OT

levels remain fairly constant in males and females over time (42 days) and higher peripheral OT

levels predict less severe symptoms, these manifest differently in each sex. For instance, female

patients with schizophrenia who have higher peripheral OT levels experience significantly

reduced delusions, hallucinations, paranoia, passive social withdrawal, and tension, and tend not

to demonstrate as much blunted affect, emotional withdrawal, and depression. There were trends

in male patients for an association between higher peripheral OT levels and lower social

avoidance and aggression. Male and female patients with higher peripheral OT levels have

significantly better prosocial scores on the Positive and Negative Symptom Syndrome Scale

(PANSS) [16].

Recent studies have shown that exogenous OT administration reduces some of the social

deficits observed in schizophrenia [17] and autism [18, 19]. In patients with schizophrenia, 3

intranasal OT significantly improved the ability to accurately identify the intentions of

individuals during a gift-giving interaction, and significantly reduced patients' positive symptoms

and overall PANSS score compared to placebo [20]. In 2010, Feifel and colleagues found that

chronic intranasal OT treatment given adjunctively with schizophrenic patients' current

antipsychotic medication significantly improved patients' total PANSS, positive symptoms, and

clinical global impressions severity (CGI-S), which measures illness severity, rate of total

improvement, and the therapeutic effect of drug treatment [21]. In a follow up study, Feifel and

colleagues further demonstrated that intranasal OT administration significantly improved

patients' total recall and performance in other cognitive and working memory tasks, as compared

to placebo [22]. In patients with autism, intranasal OT administration improved facial affect

recognition [23] and intravenous (i.v.) OT administration reduced repetitive behaviors [24] and

improved patients' ability to recognize emotion in spoken phrases [25].

Sensorimotor gating is a cognitive process that enables us to filter out irrelevant stimuli

from the constant flow of sensory and cognitive input. Deficits in sensorimotor gating are present

in patients with schizophrenia [26-30], autism [31], bipolar disorder [32, 33], obsessive

compulsive disorder (OCD) [34], attention deficit hyperactivity disorder (ADHD) [35], and are

also common in patient groups that display tic-like behavior [36, 37]. These disorders, especially

schizophrenia and autism, are associated with impairments in social processing, cognition, and

behavior, thus limiting multiple patient populations from forming typical social relationships.

Sensorimotor gating can be measured in rodents by testing prepulse inhibition (PPI) in a

sensory startle test. PPI is the suppression of the response to a startle-eliciting stimulus (such as

a loud noise or puff of air) when it is closely preceded by a less intense stimulus (such as a less

intense sound), known as the prepulse. The % PPI is the proportion of the startle response that

the subject is able to inhibit. A lower %PPI is therefore considered a worsening of sensorimotor

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gating, while a higher %PPI is considered an improvement. Previous rodent studies indicate that

OT may modulate the expression of PPI. In one study, dose-dependent OT blocked amphetamine

and dizocilpine induced deficits in PPI [38], suggesting that OT is associated with protecting

neuronal functioning in the sensorimotor gating circuit. Furthermore, PPI in Brown Norway

(BN) rats, which is regularly poor, improved after central OT administration [39]. Finally,

phencyclidine (PCP)-induced impairment of PPI was worse in OT knockout mice (compared to

wild type mice), suggesting that endogenous OT may play a role in ameliorating the PCP

induced impairment of PPI [40].

Links between deficits in social behavior and sensorimotor gating have been shown in both

disorders of cell-cell communication and in behavioral models, such as social isolation rearing.

Enzymes belonging to the Phospholipase C family (PLCs) are critical enzymes in the signal

transduction pathways of cells. and PLCs are best known for their role in cleaving

phosphatidylinositol 4,5,-bisphosphate (PIP2) into diacyl glycerol (DAG) and inositol 1,4,5-

triphosphate (IP3) in G protein-coupled receptors. Ddisruptions in PLC signaling pathways in

these processes are known to have detrimental effects on normal social behavior and

sensorimotor gating. PLC β1 KO mice showed clear PPI deficits compared to wild type mice

[41]. Socially, both sexes of PLC β1 KO mice had reduced social behavior compared to wild

type mice showing less social interaction, whisker trimming, social dominance, and nest

building. The Wnt pathway of nearly all eukaryotic organisms is a signaling pathway involved

in cell fate determination. When the Wnt protein binds to a specific G protein-coupled receptor,

the signal is sent to the Dishevelled (Dsh) protein which consists of three conserved protein

domains, one being the PDZ domain, the signal can branch off into several different pathways.

Dvl1 is a gene that codes for a Dsh that regulates cell proliferation and neuroblast specifications.

Disheveled protein found on the PDZ domain of nearly all eukaryotic organisms. PDZ domains

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are crucial for healthy cell signaling. Similar to PLC β1 KO mice, Dvl1 KO mice showed similar

deficits in social behavior and sensorimotor gating [42]. Whisker trimming, barbering, grooming,

sniffing, and nesting behavior were all reduced in Dvl1 KO mice. In male Hooded Lister rats,

social isolation rearing significantly worsened PPI at all inter stimulus intervals (the pause in

between pulses) compared to socially reared rats [43].

The dopamine system was important in restoration of PPI in both the above described cell-

cell communication models and in social isolation models [44]. For instance, PPI was restored in

PLC β1 KO mice at all prepulse levels after administration of the dopamine (D2) receptor

antagonist haloperidol [41]. In male Sprague-Dawley (SD) rats, the dopamine antagonists

seroquel [45], olanzapine [43, 45], raclopride [46], and clozapine [47] reversed the deficits in PPI

caused by social isolation rearing. Further, social isolation significantly reduced postsynaptic D2

receptor expression in the prelimbic area of the PFC, as well as worsening PPI compared to

socially reared rats [48]. In a study comparing rat strains, SD and Fischer rats, but not Lewis rats,

reared in isolation showed deficits in PPI [49] and when treated with apomorphine, a

nonselective dopamine agonist, PPI was disrupted only in healthy Fischer rats. Dopamine

agonists consistently worsen PPI in several different rodent strains [50] while dopamine

antagonists generally improve PPI, depending on the antagonist and rodent strain. This has been

demonstrated by a worsening of PPI through the administration of drugs that increase dopamine

release,with PPI restoration following administration of dopamine antagonists [51, 52].

OT has been shown to have "protective" effects against many DA agonists such as cocaine

[53, 54], amphetamine [55], and apomorphine [56, 57]. The association between DA and OT in

sensorimotor gating is unknown but anatomical studies reveal that their neuronal fibers and

receptors are close neighbors in the CNS [58, 59] as both neurotransmitter systems are essential

for producing relevant behaviors. Extensive research has revealed the importance of DA and OT

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in stimulating female and male sexual behavior, pair-bonding, and social bonding in studies of

rodents, primates, and humans [59]. Although the neuronal circuits for socioaffiliative behaviors

and sensorimotor gating are different, both behaviors are dependent on the nucleus accumbens

(NA) which is heavily populated with OT and DA receptors, specifically D2 receptors. OT and

DA in the NA facilitate partner preference [60-62] and pairbonding [63-65] in prairie voles.

Despite its efficacy as a pharmacotherapy in the abovementioned studies, it is unclear whether

OT is actually crossing the blood-brain-barrier (BBB) when administered intranasally. AVP, a

closely related neuropeptide to OT, has been shown to cross the BBB in humans [66], but OT

itself has not. OT and AVP are of a similar size, but BBB penetrance depends on many

characteristics of peptide structure. It is therefore possible that the effects of intranasal OT in

human studies are due to peripheral feedback on the central nervous system, possibly via

receptors on the myelinated vagus [67-69]. It is notable that other modes of peripheral

administration such as iv in humans [24, 25] and subcutaneous injections in voles [70] have also

been shown to affect social functioning. However, for translational reasons we felt that it was

important to examine OT effects in the mode closest to that being used in humans. Recent work

in rodents has shown that intranasal OT crosses the BBB in rodents [71] and that acute intranasal

OT at this dose results in effects on social behavior similar both in direction and effect size to

those seen in humans [72].

PPI in prairie voles has been described in only one previous study [8]. However, prairie

voles are becoming an increasingly commonly used model for drug treatments on

psychopathology [72, 73]. This preliminary study was intended to expand our knowledge of the

relationship of OT to PPI in voles. We particularly wanted to test intranasal OT at a dose both

used in humans and shown to affect social behavior acutely in voles. Antagonists were also

included as an additional probe for involvement of the OT system. This is the first study to

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investigate the effects of intranasally administered OT on PPI in rodents or tested the effects of

OT receptor antagonists (OTA). The inclusion of both an OTA that crosses the blood-brain

barrier ("central OTA" or "COTA") and one which does not ("peripheral OTA" or "POTA")

allowed us to make some initial observations as to OT’s potential site of action. We

hypothesized that intranasal OT would improve PPI as compared to placebo, and that central and

peripheral OTAs would worsen PPI as compared to placebo, with COTA reducing PPI more than

POTA.

2. Materials and methods

2.1 Experimental animals

Male (n=89) and female (n=74) prairie voles were housed with a same sex cage-mate at weaning

in standard rodent shoebox cages (27 cm x 16 cm x 13 cm) in separate rooms by sex. Animals

were housed on a 14:10-hr light-dark cycle and received food (Purina High-Fiber Rabbit chow)

and water ad libitum. Subjects were ear-clipped at least a week before initial testing. Initial

startle testing occurred between 50-60 days of age and subjects were tested a second and third

time with 7-10 days spaced between each test day. Only two males and two females from the

same litter were eligible for initial testing in order to reduce genetic bias. If neither sibling

(within each sex) demonstrated sufficient %PPI according to our cutoff detailed below, then only

one sibling was used as a subject. However, if both siblings showed sufficient %PPI, then both

were included as subjects. See Figure 1 for study design.

2.2 Startle equipment

Two SR-LAB startle boxes (San Diego Instruments, San Diego, California, USA) were used to

test PPI, with one animal per startle box. Both startle boxes contained a ventilated Plexiglas

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cylinder startle chamber (8.2 cm in diameter) mounted on a Plexiglas base (12.5 x 25.5 cm). A

speaker mounted 24 cm above the animal produced the prepulse stimuli (74, 78, 82, 86, 90 dB),

startle stimuli (120 dB), and background noise (70 dB). A piezoelectric accelerometer

underneath the base detected the animal's motion.

2.3 Startle test

Subjects underwent an initial acoustic startle test to determine PPI. Each test began with a 5

minute acclimation period with no acoustic stimuli. The acoustic startle test consisted of 7

sessions of 7 trials, where trials included five different prepulse volumes (74 dB, 78 dB, 82 dB,

86 dB, 90 dB) plus the startle; a trial containing the startle stimulus only (i.e. baseline startle);

and a trial which was a baseline reading of the animals' movement with no prepulse or startle

stimuli. These trials were presented in a pseudorandom fashion to each animal. The test

consisted of the following: prepulse for 20 ms, background noise for 80 ms, the startle stimulus

for 40 ms, background noise for 25 ms, and then no stimuli for 15 s until the next trial. % PPI

was calculated by the following formula: [1-( startle amplitude following prepulse-startle

pair/startle amplitude following startle-only)] × 100 [74]. Thus, a higher score on this measure

means more inhibition of the startle response.

We initially sorted animals into two groups dependent on response to the pre-test, using a

rough estimate of + 60% prepulse inhibition of startle. This cutoff was intended not to have any

special meaning but primarily to create two groups, one of which started with low inhibition (and

thus had room to improve when given OT); and one which started with high inhibition (and thus

had room to get worse when given an OTA). Average % PPI for the voles put into the intranasal

OT experiment was 16.08 + 9.85% at the 90dB prepulse level, while average % PPI for voles put

into the OTA experiment was 67.63 + 1.97% at the 90dB prepulse level.

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Because prairie voles generally inhibit their startle response most successfully at the loudest

prepulse tone (Bales and Palmer, unpublished data), the average score from the loudest prepulse

(90 dB) was emphasized both in the selection process and in the following analyses.

2.4 Treatments

2.4.1 Intranasal OT treatment

Animals that showed less than 60% prepulse inhibition in the initial startle test were put into the

OT study, which investigated whether intranasal OT could improve PPI. Animals (n=24)

received 25 µl of intranasal OT (0.8 IU/kg, Bachem, Torrance, CA) or saline (0.9 NaCl, Baxter,

Deerfield, IL); each animal received both treatments, one week apart. The treatments were

administered with a cannula needle attached via cannula tubing to a 23-gauge needle on a

Hamilton syringe. Treatments were administered in small droplets, alternating between nostrils.

The cannula needle did not touch the animal's nose – it was held in front of the nostril so the

animal would inhale the droplets. Saline and OT treatments were prepared prior to testing and

stored at -20°C in individual 0.6 ml microtubes. Treatment administration was randomized such

that half of the males (6 out of 12) and half of the females (6 out of 12) received intranasal OT

on their first day of treatment (second startle testing day) and the other half of the males and

females received intranasal saline. Startle testing began 15 minutes after intranasal treatments.

When administered intranasally in humans, OT has prosocial effects and is well tolerated [75].

Furthermore, our lab has found that intranasal OT administration to prairie voles is sufficient to

produce behavioral changes, similar to those seen in human studies, 15 minutes later [72].

Because the pharmacokinetics of intranasal OT are not available for prairie voles, this

therapeutic effect was used as a proxy in order to determine a reasonable dose.

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2.4.2 Intraperitoneal OTA Treatment

Animals that demonstrated over 60% PPI in the initial startle test were put into the OTA study,

which investigated whether OTA would reduce PPI. Animals (n=40, 20 males and 20 females)

received two 50 µl intraperitoneal (ip) injections. One group received either an OT receptor

antagonist with limited ability to cross the blood-brain barrier (BBB) (‘POTA’, 1 mg/kg ip,

Bachem, Torrance, CA) or saline; while the other group received either an antagonist which does

cross the BBB (‘COTA’, 1 mg/kg ip, Santa Cruz Biotechnology, Inc, Santa Cruz, CA) or saline.

Order of administration was counter-balanced. Testing began one hour post-injection for both

antagonists, based on previous studies cited below.

The POTA used, d(CH2)5[Tyr(Me2)]OVT, is an OT antagonist [76] which has been widely

used in behavioral studies in prairie voles both centrally [77] and peripherally [70, 78-80] at the

same dosage used here. This antagonist has also been shown to affect feeding behavior [81] in

rats when given peripherally, and both feeding [82] and sexual behavior [83] when given

centrally. Its molecular weight is listed as 1075.32 g/mol (Bachem, Torrance, CA), which is far

outside of the ideal range for compounds to pass through the BBB (molecular weights less than

500) [84]. However, this molecular weight is similar to that of OT (1007.2 g/mol) which crosses

the BBB only at very small percentages (1.3%) in adult rodents [85]. Please note that prairie

vole OT receptors display 92% homology with rat receptors (BLAST; accession numbers

DP001214.1 for vole OT receptor and NM_012871.2 for rat OT receptor).

The COTA used, L-368,899, is of a much lower molecular weight (554.26 g/mol) and can

penetrate the BBB in rodents [86]. It has been shown to enter the CSF and to accumulate in

limbic areas [87] in monkeys, and to affect interest in infants, sexual behavior [87] and behavior

towards pair-mates in marmoset monkeys [88], and is well-tolerated in rats and dogs [89]. For

this study, we chose the lower dosage given in [87]. While the COTA will obviously act also on

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peripheral receptors, its penetration to the central nervous system will be much higher. Although

initial doses for the two OTAs used in this project were the same, they were chosen based on

previous behavioral effects, and not intended to be at identical molar concentrations but rather to

use as a starting point for investigation of the role of the OT system in PPI.

All injections were carried out with 26-gauge needles on a Hamilton syringe. COTA and

POTA were mixed a week prior to treatments and stored at -20°C in individual 2 ml micro

centrifuge tubes. Treatment administration was also randomized. Startle testing began one hour

after ip injections based on other studies in which behavioral changes are observed one hour post

ip injection [70, 87].

2.5 Data analysis

2.5.1 Analysis of baseline and drug-tested startle amplitude

The maximum startle amplitude from the two experiments was analyzed for effects of

treatment, sex, and a treatment by sex interaction. The data of all pulse alone trials (no prepulse)

were averaged for each animal. Because animals were tested more than once, the identity of the

animal (Animal ID) was used as a random factor in the model. This prevented pseudoreplication

of results and accounted for the variability due to individual differences.

2.5.2 Analysis of % PPI

In order to simplify our models, we first checked for trial by treatment and sex by treatment

interactions. There was a significant trial by treatment interaction for the animals in the

intranasal OT experiment (F9,198 = 3.37, p = 0.0007), as well for as the animals in the OTA

experiment (F12,344 = 8.76, p < 0.0001). In the OTA experiment, there was also a trend for a sex

by treatment interaction (F2,344 = 2.78, p = 0.0631). Data were therefore analyzed separately for

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each trial, and in the OTA experiment, also analyzed separately by sex. Results for both

experiments were analyzed by mixed model ANOVA, with treatment and week as fixed effects

and animal ID as a random effect (and in the OT experiment, sex was a factor in the model as

well). Because voles display quite poor prepulse inhibition at lower prepulses, the trial with the

90 dB prepulse was viewed as the most informative trial. Residuals were checked for normality

and when necessary, transformed. Post-hoc tests were calculated using least-squares means. All

tests were two-tailed and significance was set at p < 0.05.

Because individual differences in a repeated measures design can be swamped in a standard

graph of means + standard errors, and indeed can often make significant differences look non-

significant and vice versa, the bottom panel of each graph in Figures 2-5 indicates the means +

standard errors of the difference for each individual between saline and the other treatment (OT,

COTA, POTA).

3. Results

3.1 Baseline Startle

In the intranasal OT experiment, baseline startle showed a trend for an effect of treatment (F1, 21 =

3.87, p = 0.062; Figure 2), with OT tending to result in lower baseline startle, but no significant

effect of sex or sex by treatment interaction. There was also a significant effect of animal ID

(F21, 21 = 10.78, p < 0.0001).

In the OTA experiment, baseline startle showed a significant effect of treatment (F1, 36 = 3.75, p =

0.033; Figure 3); there was no effect of sex (F1,36 = 0.02, p = 0.892), but a moderately significant

sex by treatment interaction (F2,36 = 3.23, p = 0.051). Post-hoc tests in males showed significant 13

differences between saline and COTA (t1 = 2.605, p = 0.013) and between saline and POTA (t1 =

2.177, p = 0.036). There was also a significant effect of animal ID (F38,36 = 6.84, p <0.0001).

3.2 Effects of intranasal OT administration on % PPI

For 90 dB trials, treatment had no significant effect on PPI (F1,20 = 0.49, P = 0.491; Figure 4), nor

were there any other significant predictor terms in the model. No predictor terms were significant

for trials 4, 3, or 1. However, in 78 dB trials there was a significant effect of treatment, such that

intranasal OT administration significantly worsened PPI in both sexes (F1,20 = 5.81, P = 0.026),

and a trend for animal ID (F21,20 = 1.98, P = 0.066).

3.3 Effects of intraperitoneal OTA administration on % PPI

For 90 dB trials, there was a significant effect of treatment (F2,35= 3.31, P = 0.048), with OTA

treatment disrupting PPI (Figure 5). Post-hoc tests (least-squares means) indicated that for

males, COTA significantly worsened PPI as compared to saline treatment (t = 2.57, p = 0.015),

while POTA did not (t = 0.14, P = 0.892). For females, POTA worsened PPI as compared to

saline (t = 2.11, P = 0.042), while COTA did not (t = .295, P = 0.770). Animal ID was also

significant (F38,35 = 1.94, P = 0.012).

For 86 dB trials, there were no significant terms in the model. For 82 dB trials, there was a

significant effect of animal ID (F39,35 = 2.61, P = 0.002). There were no significant effects in 78

or 74 dB trials.

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4. Discussion

This study adds to a growing literature on the role of OT receptor functioning in

sensorimotor gating. Both a centrally acting OTA (COTA), and an OTA with limited BBB

penetrance (POTA), reduced startle amplitude in males but not in females. COTA worsened PPI

significantly in males in response to a 90 dB prepulse, while females responded with worsened

PPI to the POTA. This was partially different from our expectation, that COTA would reduce

PPI more than POTA in all subjects, regardless of sex. Intranasal treatment, on the other hand,

had no effects on PPI except at 78 dB trials, in which it impaired PPI in both sexes.

Our study identified potential sex differences in how PPI is affected by the OT system in

prairie voles. Both male and female prairie voles have a high density of OT receptors in the NA

shell and core [90]. These regions of high OT receptor concentrations facilitate pair-bonding

behavior and parental care in prairie voles [91-93]. OT receptors are also present in the brain

stem and spinal cord [94, 95], areas that are heavily associated with auditory processing and

movement. The hypothesized PPI pathway in rodents includes the NA which serves as a key

relay station within the sensorimotor gating circuit [96-98]. Peripherally administered OTA (and

indeed, intranasally administered OT) may have affected baseline startle amplitude and PPI via

feedback from OT receptors in the autonomic nervous system. In prairie voles, OT has been

shown to affect heart rate variability [99], and to attenuate the autonomic response to a social

[99] or environmental [100] stressor. One possibility is that the OTAs were acting via the

myelinated vagus [69]; intranasal OT itself might also act via the trigeminal nerve [101] which

projects to the brainstem.

The Acoustic Startle Response (ASR) pathway consists of excitatory connections from

the cochlea, to the cochlear root nucleus (CRN), to the nucleus reticularis pontine caudalis

15

(NRPC), and then to the motor neurons (MN) in the spine. Within this pathway, PPI occurs

starting with excitation from the CRN and dorsal cochlear nucleus to the inferior colliculus (IC),

continues with excitation to the superior colliculus (SC) then to the pontine tegmental nucleus

(PTN), and ends with inhibition at the NRPC [102]. The limbic cortex (temporal cortex

and medial prefrontal cortex), hippocampus (HPC), ventral striatum (which

includes the NA), ventral pallidum (VP), and the pontine tegmentum

constitute the "higher" network (the "limbic cortico-striato-pallido-pontine

circuit"), helping to regulate PPI and meet with the ASR circuit at the NRPC

[102].

There is a large amount of evidence that glutamate (GLU), the most abundant

neurotransmitter in the vertebrate nervous system, is also involved in PPI. In male SD rats, PPI

was significantly worsened in social isolation reared rats after administration of the non-

competitive NMDA receptor antagonist dizocilpine (MK-80) with the highest dose disrupting

PPI even in control rats [103]. Further, pretreatment of the antipsychotics haloperidol and

clozapine failed to restore PPI after MK-80 administration [104] suggesting that perhaps DA

antagonism is not sufficient in restoring NMDA (GLU) antagonism. In another study, male SD

rats treated with MK-80 were given either the D1 agonist SKF 38393 or the D2 agonist

quinpirole to investigate the relationship between GLU and DA [105]. Treatment with SKF

38393 and dizocilpine, but not SKF 38393 or dizocilpine alone, was sufficient in disrupting PPI.

However, dizocilpine and quinpirole had no effect on PPI. Clozapine and haloperidol were

unsuccessful in restoring PPI in animals treated with dizocilpine and SKF 38393 but the D1

antagonist SCH 23390 significantly improved PPI. These results suggest that D1 receptors

support PPI if the NMDA glutamatergic system fails. GLU acts on ionotropic (NMDA, Kainate,

AMPA) and metabotropic (mGLu) receptors, as this difference in receptor type has functional 16

relevance. In DBA/2J mice, only mGlu1 receptor antagonists were able to improve PPI while

mGlu2/3, mGlu5, mGlu7, and mGlu8 antagonists had no effect [106]. Although not all mGluR

affected PPI in this study, Zou and colleagues showed that mGlu5 is involved in PPI [107].

Less is known about how central GLU and OT are associated in PPI but there is some

evidence that these systems are connected in methamphetamine administration studies. For

instance, OT inhibited the methamphetamine induced conditioned place preference in male

Swiss mice and prevented an increase in extracellular GLU in the MPFC after an

immobilization-induced stress test [108]. In a later study, OT reduced the increased expression of

NMDA receptor subunit NR1 in the PFC while increasing the expression of the GLU transporter

GLT1 in the hippocampus after methamphetamine administration [109]. Clearly OT and OTA

effects may interact with GLU and DA in order to affect startle amplitude and PPI.

Since OT receptor binding potential in the NA and other areas of the proposed PPI

pathway does not differ between male and female virgin voles [91, 93], the sex difference we

found in PPI response to the central and peripheral OTAs likely results from a mechanism other

than sex differences in central receptor binding. It is notable that male and female prairie voles

have been found to respond differently to peripherally administered OT in other contexts [70].

Previous studies of rodents have demonstrated sex differences in PPI, though the evidence is

conflicting. For instance, in Wistar rats ASR and PPI were significantly greater in males than in

females with prepulse intensities of 76, 80, and 84 dB but not 72 dB [110]. This finding is

somewhat consistent with human research in that healthy human males display greater PPI than

healthy females but at lower prepulse intensities [111]. Similarly in CBA/CaJ mice, males

showed significantly greater ASR and PPI compared to pre- but not post-menopausal female

mice [112]. Since % PPI is a calculated percentage of ASR reduction, the relationship between

17

ASR and PPI should be inversely proportional with PPI lower when ASR is greater. However, in

both these studies, this relationship was not observed.

In one study investigating the effects of estrous cycle on PPI, female SD rats showed

worsened PPI compared to males when they were in the proestrus phase of the estrous cycle, but

not when they were in the estrous or diestrus phase [113]. However, other studies have found

that the estrous cycle does not affect PPI in either Sprague-Dawley rats or in the inbred mice

strains C57BL/6J (C57) and C3H [114, 115]. Humans also show sex differences in PPI which

vary according to the menstrual cycle [116]. For instance, in the midluteal phase, when estrogen

and progesterone are highest, healthy women show the greatest reductions in PPI compared to

when they are in the follicular phase [117].

Sex differences in humans also exist with respect to the occurrence and severity of

schizophrenia. Specifically, schizophrenia is more prevalent and deleterious in males than in

females [118], and there is evidence that menstrual cycle affects the course of the disorder.

Although plasma OT levels do not change during the menstrual cycle in women with

schizophrenia, social cognition and overall psychopathology vary during different phases of the

cycle [119, 120]. It is possible that estrogen is primarily responsible for this variation since both

the genes for OT and OT receptors are estrogen responsive [121, 122].

The fact that intranasal OT had no significant effect on improving PPI, and even

significantly worsened PPI at 78 dB, was unexpected based on previous studies [123], especially

when considering that the circuits involved in social behavior and those involved in PPI share

some common areas. It is notable that OT at this dosage also tended to reduce baseline startle

amplitude. One possible explanation for this unforeseen result is that the intranasal OT dosages

which acutely increase pro-social behavior in prairie voles [124] differ from those necessary to

alter PPI. A complete dose-response study of intranasal OT would help distinguish which

18

dosages are optimal for improving social behavior and PPI, and the extent to which they differ.

This could then become an important treatment consideration when translating to human

subjects. In addition to dosage, our chosen route of OT administration and/or the time period in

between OT treatment and startle testing could have been misguided which might explain why

PPI was unaffected after intranasal OT treatment. In the two studies looking at OT and PPI in

rats, OT was administered subcutaneously and startle testing began 30 min after OT treatment

[123]. However, startle testing began 10 min after subcutaneous OT administration when it was

quickly followed by subcutaneous amphetamine, apomorphine, and MK-801 administration [55]

which suggests that peripheral OT may influence startle testing more than central OT. If we had

included a test group that received peripheral OT with known working dosages [125, 126], then

we could compare PPI from the intranasal and peripheral OT groups. Another test group we

could have included is a group that received a DA antagonist for positive control. In all of the

existing literature the authors included application of drugs manipulating the DA system and so it

may have been wise to apply DA antagonists to the poorly inhibiting voles and see if this

principle holds true...

Another potential reason why OT did not significantly improve PPI might be due to

differences between inbred versus out-bred rodents. OT administered subcutaneously to male

Sprague-Dawley (SD) rats, which are out-bred, had no significant effect on PPI from baseline.

However, in Brown Norway (BN) rats, which are in-bred, increases in the dose of subcutaneous

OT injections were directly related to increases in % PPI of the startle response [123]. The

prairie voles used in our study were out-bred and it is possible that inbred strains have linked

genes associated with OT receptor morphology in the PPI circuitry. Previous reports on PPI and

antipsychotics in rodent studies indicate that there are differences in drug effectiveness based on

inbred and out-bred strains [127-129]. In this case, we might expect an out-bred rodent species to

19

be the best translational model for humans. The prairie voles in our study showed considerable

individual differences, shown by the significance of the random factor “ID”, which could relate

to baseline differences in OT, DA, or GLU receptors, and which make an interesting topic for

further study.

There were several limitations of this study, which was intended to be preliminary in

many respects. For both OT and OTAs, we used only single dosages without dose-responses.

While this weight-adjusted dose of OT has been shown to cause similar acute changes in social

behavior in voles as it does in humans [72], the differing construction of the rodent nose and

differences in potential modes of administration (which can vary or be poorly described even in

many human studies) may also have affected the current findings [130]. In addition to more

doses, it would be useful to have combined agonist/antagonist treatments, as well as an

antipsychotic drug treatment as a positive control. Because this study uses a different species as

well as a different route for OT treatment, additional experiments are required to compare

directly to results from rat and mouse models.

In summary, we found that while intranasal OT had minimal or detrimental effects on

PPI at the dosage used to increase social contact in this species, male PPI responded to a

centrally acting OTA and female PPI to a peripherally acting OTA. Full dose-responses for both

OT and OTAs will be needed to confirm and extend these very preliminary results; however,

these results suggest that further investigation of the role of OT in the PPI circuit is warranted.

5. Acknowledgements

This research was supported by National Institutes of Child Health and Human Development

grants 071998 and 060117, and the University of California, Davis. We would like to thank

Allison Perkeybile for general assistance, Benjamin Ragen and Sara Freeman for assistance in

20

editing of the manuscript, and Drs. Rhonda Oates-O’Brien and Cindy Clayton for veterinary

assistance and animal care.

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Figure Legends.

Figure 1. Study design. Subjects were sorted into two groups based on a initial pre-test.

Animals displaying 60% or greater PPI were put into the OTA experiment (left half of figure),

while animals displaying poor inhibition (less than 60% PPI) were put into the OT experiment

(right half of figure). Average %PPI for the voles put into the intranasal OT experiment was

16.08 + 9.85% at the 90dB prepulse level, while average %PPI for voles put into the OTA

experiment was 67.63 + 1.97% at the 90dB prepulse level. Each animal received two treatments

(saline and OT; saline and COTA; or saline and POTA) in a counter-balanced fashion.

Figure 2. Baseline startle in the intranasal OT study. Startle amplitude in pulse only trials in the

OT study showed a trend for animals given OT to display lower startle amplitude (F1, 21 = 3.87, p

= 0.062). The top panel shows means and standard errors by group. Because individual

differences in a repeated measures design can be swamped in a standard graph of means +

standard errors, the bottom panel indicates the means + standard errors of the difference for each

individual between saline and OT treatment (thus a positive difference means startle amplitude

was higher when given saline).

Figure 3. Baseline startle in the OTA study. Startle amplitude in pulse only trials in the OTA

study showed a significant effect of treatment (F1, 36 = 3.75, p = 0.033) and a moderately

significant sex by treatment interaction (F2,36 = 3.23, p = 0.051). Post-hoc tests in males showed

significant differences between saline and COTA (t1 = 2.605, p = 0.013) and between saline and

POTA (t1 = 2.177, p = 0.036). Because individual differences in a repeated measures design can

be swamped in a standard graph of means + standard errors, the bottom panel indicates the

28

means + standard errors of the difference for each individual between saline and OTA treatment

(thus a positive difference means startle amplitude was higher when given saline). Significant

differences are marked by asterisks.

Figure 4. %PPI in the intranasal OT study. In 78 dB trials of intranasal OT treatment there was a

significant effect of treatment, such that intranasal OT administration significantly worsened PPI

in both sexes (F1,20 = 5.81, P = 0.026). Because individual differences in a repeated measures

design can be swamped in a standard graph of means + standard errors, the bottom panel

indicates the means + standard errors of the difference for each individual between saline and OT

treatment (thus a positive difference means %PPI was higher when given saline). Significant

differences are marked by asterisks.

Figure 5. % PPI in the OTA study (males). In 90 dB trials of OTA administration, there was a

significant effect of treatment (F2,35= 3.31, P = 0.048), with OTA treatment worsening PPI. Post-

hoc tests (least-squares means) indicated that for males, COTA significantly worsened PPI as

compared to saline treatment (t = 2.57, p = 0.015), while POTA did not (t = 0.14, P = 0.892).

Because individual differences in a repeated measures design can be swamped in a standard

graph of means + standard errors, the bottom panel indicates the means + standard errors of the

difference for each individual between saline and OTA treatment (thus a positive difference

means %PPI was higher when given saline). Significant differences are marked by asterisks.

29

Figure 6. % PPI in the OTA study (females). For females, POTA worsened PPI as compared to

saline (t = 2.11, P = 0.042), while COTA did not (t = .295, P = 0.770). Because individual

differences in a repeated measures design can be swamped in a standard graph of means +

standard errors, the bottom panel indicates the means + standard errors of the difference for each

individual between saline and OTA treatment (thus a positive difference means %PPI was higher

when given saline). Significant differences are marked by asterisks.

30