Obsessive-compulsive-like behaviors in house mice are attenuated by a probiotic (Lactobacillus rhamnosus GG).

Research report 71

Obsessive–compulsive-like behaviors in house mice are attenuated by a probiotic (Lactobacillus rhamnosus GG) Pranish A. Kantak, Dylan N. Bobrow and John G. Nyby Two experiments examined probiotic pretreatment (Lactobacillus rhamnosus GG) on obsessive–compulsive disorder (OCD)-like behavior induction by RU 24969 in BALB/cJ house mice. In the first experiment, two groups were defined by their daily pretreatment by oral gavage of either (a) L. rhamnosus (1 ¾ 109 CFU/day) or (b) the saline vehicle. Both a 2- and 4-week probiotic pretreatment attenuated OCD-like behavior induction (increased perseverative open-field locomotion, stereotypic turning, and marble burying) relative to saline pretreatment. Experiment 2 re-examined the 2-week probiotic pretreatment while also comparing it to a 4-week fluoxetine pretreatment. Again, groups were defined by daily pretreatment of either (a) L. rhamnosus for 2 weeks, (b) the saline vehicle for 2 weeks, or (c) fluoxetine (10 mg/kg) for 4 weeks. Pretreatment by either L. rhamnosus or fluoxetine blocked the induction of OCD-like behavior compared with saline pretreatment. Thus the

2-week probiotic pretreatment was again effective. Although side effects of fluoxetine or L. rhamnosus on androgen-dependent behaviors could not be demonstrated, L. rhamnosus treatment appeared comparable to fluoxetine treatment in attenuating mouse OCD-like behaviors. c 2014 Wolters Behavioural Pharmacology 25:71–79 Kluwer Health | Lippincott Williams & Wilkins.

Introduction

anxiety-like measures in mice, whereas Messaoudi et al. (2011) similarly found that probiotic treatment reduced anxiety measures in rats and humans. Messaoudi et al. (2011) also found that probiotic-treated humans scored lower on the obsessive–compulsive subtest on the Hopkins Symptoms Checklist-90, providing preliminary evidence for probiotic efficacy in treating human OCD.

Obsessive–compulsive disorder (OCD) is an anxiety disorder over which a sufferer has little control, characterized by obsessive anxiety-producing thoughts often alleviated by compulsive behaviors (Jones et al., 2012). Individuals with OCD often go undiagnosed and untreated. More common than originally thought, OCD has a lifetime and 12-month prevalence in the USA of 2.3 and 1.2%, respectively (Ruscio et al., 2010). Imbalances in central serotonergic (5-HT) activity are widely thought to play a causative role in the etiology of OCD (Karch and Pogarell, 2011; Angoa-Perez et al., 2012). In support of this hypothesis, serotonin reuptake inhibitors (SRIs), such as fluoxetine (Prozac), are the first-line drug therapy for OCD, and the only approved monotherapy, despite effectiveness in only 40–60% of patients. Because of inconsistent benefit and significant side effects [i.e. sexual dysfunction, headaches, and jitteriness (Storch and Merlo, 2006; Fagiolini et al., 2012)], better pharmacotherapies are desirable. In our study, we examined whether Lactobacillus rhamnosus GG (a probiotic bacterium) would alleviate OCD-like behaviors in mice. Probiotic organisms clearly provide digestive benefit, and have proven value in treating gastrointestinal (GI) disorders such as irritable bowel syndrome and diarrhea (Desbonnet et al., 2010). More relevant to our hypothesis, Bravo et al. (2011) found that chronic probiotic treatment reduced depressive and c 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins 0955-8810

Behavioural Pharmacology 2014, 25:71–79 Keywords: fluoxetine, Lactobacillus rhamnosus, locomotor activity, mice, OCD, open field, perseveration, probiotics, RU 24969, SRI Department of Biological Sciences, Lehigh University, Bethlehem, Pennsylvania, USA Correspondence to John G. Nyby, PhD, Department of Biological Sciences, 111 Research Drive, Lehigh University, Bethlehem, PA 18015, USA E-mail: [email protected] Received 11 February 2013 Accepted as revised 18 October 2013

In our study, OCD-like behaviors were induced using RU 24969 (a 5-HT1A/1B receptor agonist). Supporting the validity of this approach, Shanahan et al. (2011) found that SRI pretreatment attenuated RU 24969 induction of mouse OCD-like behaviors. Furthermore the required treatment time frame (4 weeks of SRI pretreatment) was comparable to that used in treating human OCD. In further support of the RU 24969 model, 5-HT1B receptor agonists (such as RU 24969) also exacerbate OCD symptoms in humans [reviewed by Shanahan et al. (2011)]. Two experiments examined whether pretreatment with a probiotic bacterium, L. rhamnosus GG would prevent OCD-like symptoms in mice caused by RU 24969 injection. The effectiveness of L. rhamnosus was also compared with that of fluoxetine. To assess OCD-like behaviors, we measured various parameters of open-field locomotion including distance traveled, amount of time spent in movement, distance traveled per movement, thigmotaxis, stereotypical center-point turns, and beam breaks accompanied by little body movement. DOI: 10.1097/FBP.0000000000000013

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72 Behavioural Pharmacology 2014, Vol 25 No 1

As an additional measure of perseverative behavior, we also measured marble burying (Thomas et al., 2009). To assess possible reproductive side effects, we measured two androgen-dependent behaviors: intermale aggression and precopulatory ultrasonic calling. In experiment 1, both 2 and 4 weeks of probiotic pretreatment were effective in attenuating the induction of OCD-like behaviors by RU 24969. In contrast, Shanahan et al. (2009) found that while 4 weeks of fluoxetine pretreatment attenuated OCD-like behavior induction, 2 weeks did not. Thus, experiment 2 examined whether the effectiveness of 2 weeks of probiotic pretreatment could be replicated. In addition, we compared the 2-week probiotic pretreatment with an effective fluoxetine pretreatment (4 weeks of pretreatment) and also examined two androgen-dependent male-typical behaviors to examine possible side effects of fluoxetine and probiotics; intermale aggression and 70 kHz reproductive calling.

Methods Subjects and housing

Male Balb/cJ mice (Jackson Laboratories, Bar Harbor, Maine, USA), 8 weeks of age and weighing 20–25 g, served as subjects. This strain was selected because OCD-like behaviors were previously inducible with the serotonin agonist, RU 24969. In addition, this strain responds well to SRI treatment in blocking the OCD-like effects of RU 24969 (Dulawa et al., 2004; Shanahan et al., 2011). We also selected males to examine possible treatment side effects upon two androgen-dependent, male-typical behaviors: intermale aggression and 70 kHz ultrasonic calling to females. As group-housed male Balb/cJ mice exhibit excessive fighting (Shanahan et al., 2011), subjects were individually housed upon arrival in opaque plastic cages (28 18 12 cm3) with metal cage tops. The colony room was maintained at a controlled temperature (20 ± 21C) in the Lehigh University Central Animal Facility on a 14/10 light/dark cycle with free access to food and water. All pretreatments, injections, and behavioral testing occurred between 14:00 and 18:00 h. The Lehigh University Institutional Animal Care and Use Committee approved the protocol. Adult male and female CD-1 mice (Charles River Laboratories, Malvern, Pennsylvania, USA), a standard outbred strain used in other behavioral research in our laboratory, served both as social-experience animals and as stimuli in the aggression and vocalization tests. Probiotic preparation

L. rhamnosus, a common bacterial strain used to treat GI disorders in humans (Guandalini, 2011), was used. This strain also has probiotic effects in mice (Gill and Rutherfurd, 2001; Zhang et al., 2013).

A freeze-dried pellet of L. rhamnosus was obtained from the American Type Culture Collection (Manassas, Virginia, USA) (ATCC 53103) and inoculated into fresh Lactobacilli MRS broth (BD 288130). The time course for bacterial growth was assessed using a bacterial plate counter and a 600 nm optical-density curve plotted. The culture reached the desired concentration of 1 109 colony forming units (CFU) at 40 h postinoculation. Aliquots (400 ml) were then transferred to microcentrifuge tubes and spun down for 2 min at 13 000 rpm. The supernatant was drawn off and the pellet resuspended in 200 ml of physiological saline solution. From this suspension 100 ml was drawn off for oral gavage. Probiotic and fluoxetine pretreatment

The dosages of fluoxetine and L. rhamnosus selected were within the effective range used in other published research. For example, fluoxetine (Fisher Scientific, Pittsburgh, Pennsylvania, USA), at 10 mg/kg/day was effective in blocking OCD-like behaviors in mice (Shanahan et al., 2011). L. rhamnosus at a density of 1 109 CFU/day was within the ranges protecting against rotavirus-induced diarrhea in neonatal mice (Zhang et al., 2013), and antagonizing oxidative stress and intestinal disaccharidases in giardia-infected Balb/cJ mice (Goyal et al., 2013). Both treatments were administered daily in 0.1 cc physiological saline by oral gavage, using reusable curved metal animal feeding needles (3.81 cm length; 2.25 mm ball diameter) (Cadence Life Science, Staunton, Virginia, USA). Needles were autoclaved between treatments. As we only utilized a single daily concentration of L. rhamnosus, future research will be necessary to establish the range of effective daily probiotic dosages. Induction of obsessive–compulsive disorder-like behavior

RU 24969 (Tocris Bioscience, Minneapolis, Minnesota, USA) was dissolved in saline and acutely injected intraperitoneally, at a concentration of 10 mg/kg, to induce OCD-like behaviors. Behavioral testing began 60 min after RU 24969 injection. This time frame was chosen based upon the work of others (Clement et al., 2009), as well as preliminary pilot work. Behavioral testing Social-experience regimen

Before beginning behavioral testing, all subjects underwent a social-experience regimen to optimize the occurrence of male-typical reproductive behaviors (Nyby, 2009). For 8 consecutive days, a male and female stimulus animal were sequentially placed in the subject’s home cage for 3 min each. An interval of about 10 min intervened between presentations, and the sex of the stimulus presented first alternated each day. When aggression occurred toward the male stimulus (as progressively


Probiotics and OCD Kantak et al. 73

occurred over the days), the stimulus was removed immediately to minimize injury. Aggression to females was not seen. Behavior measurement and sequencing

Behavioral measurement by a human observer (marble burying, vocalizations, aggression) was performed single blind to minimize observer bias. All open-field measurements were computer scored.

used in the treatment of OCD (Deacon, 2006). However, we do acknowledge that marble burying also responds to some drugs not commonly used to treat human OCD (Deacon, 2006) and that some caution should be used in extrapolating to human OCD.

Half the subjects in each treatment group were tested first in the open field and half in marble burying. An interval of B20–25 min intervened between tests. In experiment 2, after completing these tests, reproductive motivation (70 kHz ultrasonic calling) and intermale aggression were subsequently examined.

To measure marble burying, an opaque plastic cage (32 22 14 cm3) containing 5 cm of clean bedding served as the test chamber. Twenty-four hours before testing, subjects were habituated to the chamber for 30 min. The following day, beginning either 60 or 85 min following RU 24969 injection, the subjects were placed in the chamber now containing 15 evenly spaced marbles (3 5 matrix). After 30 min, the subject was removed and the number of buried marbles counted. A buried marble was defined as 2/3 buried (Deacon, 2006).

Open field

Ultrasonic vocalizations

Beginning either 60 or 85 min after RU 24969 administration, subjects were placed in the center of a 40 40 40 cm3 TruScan open-field chamber made of clear Plexiglas (Coulbourn Instruments, Allentown, Pennsylvania, USA) for 20 min to assess locomotor behaviors. The chamber was open at the top with a removable Plexiglas floor. A 32 32 photobeam sensor ring surrounding the bottom of the chamber accurately monitored subject location using photobeam occupation averaging. Six subjects were measured concurrently in six available TruScan chambers.

The microphone of a QMC Ultrasonic Receiver (QMC, London, UK), tuned to a center frequency of 70 kHz, was placed B15 cm above the subject’s home cage. Before stimulus presentation, the subject was monitored for ultrasonic calling for 1 min to ensure that premature calling did not occur. No calling occurred during this habituation period. The 3-min test began by placing a female in the subject’s home cage. The number of 5-s time-sampling intervals containing calling reflected the amount of ultrasonic calling, with possible scores ranging from 0 to 36.

TruScan software (Coulbourn Instruments) calculated the following measures of locomotor activity: (a) distance traveled in centimeters, (b) amount of time spent moving in seconds, (c) distance traveled per move, reflecting perseverative locomotion, (d) distance traveled in the center of the open field, reflecting the inverse of thigmotaxis, (e) total stereotypic turns (sum of stereotypic clockwise and counterclockwise turns around a single point), and (f) nonmotile beam breaks (labeled STPY-1 in TruScan Software). Each instance of STPY-1 consisted of three or more repetitive movements with intermovement intervals of less than 2 s when the geometric center of the mouse moved less than the distance between adjacent beams. We interpreted this measure as reflecting movement hesitancy (the opposite of movement perseveration). Between tests, the floors were removed and cleaned with soap and water and the Plexiglas walls wiped with a wet sponge. Marble burying

Marble burying was measured as an ‘OCD-like’ behavior for two reasons. Thomas et al. (2009), surveying 10 different inbred mouse strains, provided ecological validity by concluding that mouse marble burying is a very useful tool for examining genetically determined repetitive/perseverative behaviors. In addition, marble burying also responds to the same SRIs and anxiolytics

Ultrasonic vocalizations are an androgen-dependent behavior reflective of male sexual arousal (Nyby, 2009). Females do not normally call in male/female pairings. Thus any ultrasounds detected could be attributed almost exclusively to the male (White et al., 1998). Upon completion of the test, the female was removed from the male’s home cage. Intermale aggression

Within 1 min after completing the ultrasonic calling test, a novel male was introduced into the subject’s home cage and the subject’s latency to attack was recorded by an experimenter ‘blind’ to subject identity. The test was terminated upon first attack or after 3 min had elapsed. Intermale aggression is also androgen dependent. Experiment 1

Following the social-experience regimen, 18 male Balb/cJ mice were assigned to one of two treatment groups: (a) L. rhamnosus gavage (n = 12) or (b) saline gavage (n = 6). Following 2 weeks of pretreatment and again following 4 weeks of pretreatment, subjects received an intraperitoneal injection of RU 24969 (10 mg/kg) to induce OCD symptoms. One hour following RU 24969 injection, half the subjects in each group were tested first in the marble burying test and half tested first in the open field. Upon completion of the first test (B20 min


74 Behavioural Pharmacology

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later), the subjects were immediately tested in the other behavioral test.

Fig. 1

One 2-week probiotic animal, two 4-week probiotic animals, and four 4-week fluoxetine animals were excluded from behavioral testing because of injection errors. In addition one 4-week fluoxetine animal was excluded from statistical analysis because of its status as an outlier on various locomotor measures (more than 2 SDs from the group mean). Statistical analysis

All data were analyzed using analysis of variance (ANOVA; SPSS 18; SPSS Inc., Chicago, Illinois, USA; site-licensed to Lehigh University). Experiment 1 utilized a 1-between (groups) 1-within (2- vs. 4-week pretreatment) factor ANOVA, whereas experiment 2 utilized a 1-factor between-groups ANOVA. In experiment 2, selected between-subject contrasts utilized Tukey post-hoc tests.

Results Experiment 1 Locomotor behavior

As seen in Fig. 1, RU 24969 injection produced similar behavioral results after 2 and 4 weeks of pretreatment. In general, probiotic-pretreated subjects traveled significantly less distance [Fig. 1a: F(1, 15) = 36.4; P < 0.001] and spent significantly less time in movement [Fig. 1b: F(1, 15) = 49.6; P < 0.001] than the saline-pretreated subjects. Reflecting their greater movement perseveration, the saline-pretreated subjects traveled significantly farther per movement [Fig. 1c: F(1, 15) = 52.0; P < 0.001]. As seen in Fig. 2, group differences in other movement parameters were also observed. Again, no effect of duration of probiotic pretreatment was observed. In general, saline-pretreated subjects were less thigmotactic, as reflected in their traveling a greater distance in the center of the apparatus [Fig. 2a: F(1, 15) = 8.0; P < 0.02]. The saline-pretreated also made more stereotypic turns during a movement [Fig. 2b: F(1, 15) = 57.2; P < 0.001]. The saline-pretreated animals also showed fewer nonmotile beam breaks [Fig. 2c: STPY-1: F(1, 15) = 52.0; P < 0.001], which we interpret as reflecting their lesser movement hesitancy.

Probiotic

Saline

17 000 15 000 13 000 11 000 9000 7000 5000

(b) Mean movement time (s)

Four groups were defined by their pretreatment before OCD-like behavior induction: (a) 2-week probiotic (n = 12), daily gavages of L. rhamnosus for 2 weeks; (b) 4 week fluoxetine (n = 12), daily gavages of fluoxetine for 4 weeks; (c) 2 week control (n = 6), daily saline gavages for 2 weeks; and (d) 4 week control (n = 6), daily saline gavages for 4 weeks. As the two saline-treated control groups responded similarly to RU 24969, they were combined for graphical presentation and statistical analysis.

(c) Mean distance per move (cm)

Experiment 2

Mean distance traveled (cm)

(a)

1200 1150 1100 1050 1000 950 900 850 800 750 700 250 200 150 100 50 0

2 weeks 4 weeks Length of pretreatment

Behaviors in an open field [(a) mean distance traveled, (b) mean move time, (c) mean distance per move] seen in a 20-min test by subjects pretreated by oral gavage with either a probiotic (Lactobacillus rhamnosus) or saline. OCD-like behaviors were induced with RU 24969 following 2 and 4 weeks of pretreatment. OCD, obsessive–compulsive disorder.

Marble burying behavior

As seen in Fig. 3, the saline-pretreated subjects buried significantly more marbles than the probiotic-pretreated subjects [F(1, 16) = 150.24; P < 0.001]. Experiment 2 Locomotor behavior

There was a trend for the saline-pretreated animals to travel farther in the 20-min open-field test (Fig. 4a), although the overall difference among the groups missed our P < 0.05 criteria for significance [F(2, 25) = 3.372;



Fig. 3

Probiotic


(a)

Saline

9000 8000 7000 6000 5000 4000

Mean percentage of marbles buried

Fig. 2

3000

100 90 80 70 60 50 40 30 20 10 0

Probiotic Saline

2 weeks

2000

Length of pretreatment Number of marbles buried with cage shavings in a 30-min test by subjects pretreated by oral gavage with either a probiotic (Lactobacillus rhamnosus) or saline. OCD-like behaviors were induced with RU 24969 following 2 and 4 weeks of pretreatment. OCD, obsessive–compulsive disorder.

(b) 165 145 125 105

Fig. 4

85 (a) Mean distance traveled (cm)

65 45 25

500 400 300

100 0

13 000 12 000 11 000 10 000 9000 8000 7000

(b)

200

2 weeks 4 weeks Length of pretreatment

Behaviors in an open field [(a) distance traveled in the center, (b) stereotypic turns, (c) nonmotile beam breaks] seen in a 20-min test by subjects pretreated by oral gavage with either a probiotic (Lactobacillus rhamnosus) or saline. OCD-like behaviors were induced with RU 24969 following 2 and 4 weeks of pretreatment. OCD, obsessive–compulsive disorder.

P = 0.051]. Nor were the selected contrasts (Tukey test) of the probiotic or fluoxetine groups with the control significant. However on all other measures of locomotor activity, the saline-pretreated animals were more active. Significant overall group differences were seen in move time [Fig. 4b: F(2, 25) = 6.34; P < 0.01], and in distance per move [Fig. 4c: F(2, 25) = 4.70; P < 0.02] reflecting the more perseverative locomotion of the saline-pretreated controls. Group differences were also seen in other movement parameters. For example the groups differed significantly

Mean movement time (s)

Mean number of beam breaks

600

1100 1050 1000 950 900 850 800

(c) Mean distance per move (cm)

Mean number of turns

185

(c)

4 weeks

210 190 170 150 130 110 90 70 50 Saline

Probiotic 2 weeks Fluoxetine 4 weeks Type of pretreatment

Behaviors in an open field [(a) mean distance traveled, (b) mean move time, (c) mean distance per move] seen in a 20-min test by subjects pretreated by oral gavage with either a probiotic (Lactobacillus rhamnosus), fluoxetine, or saline. OCD-like behaviors were induced with RU 24969 following 2 weeks of pretreatment. OCD, obsessive–compulsive disorder.



Amount of ultrasonic calling to a female stimulus and latency to aggression toward a male stimulus during 3-min tests

Fig. 5

Table 1


(a) 7250 6750 6250 5750 5250 4750 4250 3750 3250 2750 2250

Mean number of turns

(b)

Behavior Amount of ultrasonic calling Aggression latency (s)

Saline (2 weeks)

Probiotic (2 weeks)

Fluoxetine (4 weeks)

0±0 52.73 ± 2.91

0±0 24.19 ± 4.14

0±0 35.33 ± 6.45

The subjects were pretreated by oral gavage with either a probiotic (Lactobacillus rhamnosus) or saline for 2 weeks or fluoxetine for 4 weeks and then injected with RU 24969 B 90 min before behavioral testing.

in the distance traveled in the center of the apparatus [Fig. 5a: F(2, 25) = 3.92; P < 0.05], reflecting the greater thigmotaxis of the probiotic-pretreated and fluoxetinepretreated subjects. The groups also differed in the number of stereotypic turns made during a movement [Fig. 5b: F(2, 25) = 4.4; P < 0.03], reflecting the greater number of turns made by the saline-pretreated subjects, and in nonmotile beam breaks [Fig. 5c: F(2, 25) = 5.63; P < 0.02], which we interpret as also reflecting the greater movement perseveration of the saline-pretreated subjects.

145 125 105 85 65 45 25

Mean number of beam breaks

(c)

Marble burying 500

As seen in Fig. 6, the groups differed in amount of marble burying [F(2, 25) = 10.68; P < 0.001] reflecting the finding that the saline animals buried more marbles than subjects from the other two treatment groups.

450 400 350 300

Intermale aggression and 70 kHz ultrasonic calling

250 200 Saline


Behaviors in an open field [(a) distance traveled in the center, (b) stereotypic turns, (c) nonmotile beam breaks] seen in a 20-min test by subjects pretreated by oral gavage with either a probiotic (Lactobacillus rhamnosus), fluoxetine, or saline. OCD-like behaviors were induced with RU 24969 following 2 weeks of pretreatment. OCD, obsessive–compulsive disorder.

Discussion

Fig. 6

Mean percentage of marbles buried

As seen in Table 1, no males from any of the groups emitted ultrasounds to the female stimulus. Thus the groups were clearly not different on this measure. Subjects from all treatment groups were highly aggressive and attacked the intruder male almost immediately. Although the saline subjects appeared less aggressive (took longer to attack) than the probiotic and fluoxetine subjects, the overall group difference was not statistically significant [F(2, 25) = 1.43; NS].

70.00 65.00 60.00 55.00 50.00 45.00 40.00 35.00 30.00 25.00 20.00 Saline


Number of marbles buried with cage shavings in a 30-min test in mice pretreated by oral gavage with either a probiotic (Lactobacillus rhamnosus), fluoxetine, or saline. OCD-like behaviors were induced with RU 24969 following 2 weeks of pretreatment. OCD, obsessive–compulsive disorder.

Our major novel finding was that both 2 and 4 weeks of pretreatment with L. rhamnosus GG attenuated the induction of OCD-like behaviors in mice. To our knowledge this is the first demonstration that a probiotic can reduce OCD-like behaviors in an animal model. Although probiotic pretreatment appeared to act more quickly than previously found for fluoxetine (Shanahan et al., 2009) methodological differences could account for the difference (i.e. elapsed time following RU 24969 injection, sex differences, differences in social/sexual experience: see below). As our OCD-like behaviors all involved accentuated locomotion, we cannot entirely rule out the possibility that our probiotic pretreatment exerted nonspecific effects upon the behaviors tested (perhaps by making the animals sick or sedating them). However, for a



number of reasons, we believe these explanations unlikely. Although we did not quantitatively measure the health of our mice, subjectively we did not observe any obvious effects upon their health or body weight. Moreover, many published studies find that chronic administration of L. rhamnosus generally promotes good health in mice when their health is challenged by experimental manipulations. For example, chronic administration of L. rhamnosus improved the immune status of immunocompromised mice (Wei et al., 2007) and the insulin sensitivity of mice fed a high-fat diet (Kim et al., 2013). Body weight was not adversely affected by L. rhamnosus administration in either of these studies, further supporting the idea that L. rhamnosus does not cause illness. We were unable to find published research addressing possible sedative effects of L. rhamnosus in mice. However, based upon research we describe later, we believe our probiotic effects were most likely mediated through effects upon OCD-relevant neural circuitry. Our findings replicated previously published work (Shanahan et al., 2009, 2011) showing that an acute injection of RU 24969 induced OCD-like behavior in mice and that 4 weeks of fluoxetine pretreatment attenuated this effect. We found that RU 24969 enhanced distance traveled by saline-pretreated controls mainly by increasing the time spent, and distance covered, per movement (i.e. perseverative hyperlocomotion). These behaviors were counteracted by both fluoxetine and probiotic pretreatments. Probiotic-pretreated and fluoxetine-pretreated subjects also displayed enhanced levels of an open-field behavior (nonmotile beam breaks) that we interpret as movement hesitancy. We believe that the perseverative hyperlocomotion of the saline-pretreated controls was related to their low levels of movement hesitancy. Perseverative marble burying was also reduced similarly by fluoxetine and probiotic pretreatment. Thus, our results support previous literature (Shanahan et al., 2009, 2011) correlating these rodent behaviors to the compulsive behaviors exhibited by humans with OCD. However, one locomotor difference from the results reported by Shanahan et al. (2011) was the effect of RU 24969 on open-field thigmotaxis. Although Shanahan et al. (2011) found greater thigmotaxis, we found the opposite. However, methodological differences likely account for the difference. Although we both used Balb/cJ mice from Jackson Laboratories, Shanahan et al. (2011) used female subjects while we used males, suggesting the possibility of a sex difference in the effects of RU 24969 upon thigmotaxis. Alternatively, our 8-day social-experience regimen before testing could have contributed to the different outcome, as well as our greater elapsed time between RU 24969 injection and behavioral testing. Our impressions were that the enhanced locomotion of our saline-pretreated subjects made them more indis-

criminate in their open-field movements while the probiotic-pretreated and fluoxetine-pretreated subjects displayed the thigmotaxis more characteristic of untreated mice. One side effect of fluoxetine observed in humans and rats is a reduction of sexual motivation (Vega Matuszcyk et al., 1998), perhaps resulting from effects on sex-hormone signaling. Consequently, we examined two androgendependent male-typical behaviors; 70 kHz ultrasonic calling to female stimuli (reflective of sexual motivation) and intermale aggression (reflective of aggressive motivation). However, none of our male subjects emitted calls so we were unable to test the hypothesis with this behavior. In retrospect, a strain survey (Nyby and Whitney, 1978) found BALB/cJ males not to be robust callers so maybe another strain that calls more reliably and with shorter latency would allow a more valid test of the hypothesis. Although the saline-pretreated subjects appeared less aggressive than the probiotic-pretreated and fluoxetinepretreated subjects following RU 24969 injections, the effect was not statistically significant. Our failure to find a pretreatment effect on intermale aggression may have been due to a ‘ceiling effect.’ Balb/cJ males are inherently highly aggressive toward other males (Shanahan et al., 2011). Utilizing a different strain with a lower aggression baseline might produce different results. However, our results are consistent with a growing number of findings demonstrating that probiotic activity in the gut influences central nervous system functioning and mood (Grenham et al., 2011; Cryan and Dinan, 2012; Saulnier et al., 2013). For example, Huang and Reichardt (2001) found that germ-free mice lacking gut bacteria had reduced levels of brain-derived neurotrophic factor, and Neufeld et al. (2011) found decreased levels of 5-HT1A mRNA expression in the dentate gyrus. In addition, compared with conventionally colonized subjects, Clarke et al. (2013) found that germ-free mice had an exaggerated stress response, characterized by increased corticosterone and tumor necrosis factor-a in response to an acute stressor. Various lines of evidence using mice implicate the vagus nerve as a likely communicatory channel by which gut probiotic activity affects the brain. For example, Lyte et al. (2006) found increased anxiety during the early stages of a GI bacterial infection before overt disease symptoms, suggesting that the anxiety was not caused by a systemic inflammatory response. Supporting vagal communication, the vagal afferents of infected mice also showed cFos activation. Bercik et al. (2011) found that colitis-induced anxiety in mice could be reversed either by probiotic administration (Bifidobacterium longum NCC3001) or by subdiaphragmatic vagotomy. Further supporting vagal communication, in-situ exposure of enteric neurons to probiotic fermentation products caused hyperexcitability



to depolarizing voltages (Bercik et al., 2011). Interestingly a strain of Lactobacillus spp. also enhanced GABA-receptor expression in the brain through vagal communication in mice (Bravo et al., 2011). Very recently, Tillisch et al. (2013) provided the first evidence that probiotics affect brain functioning in normal healthy human females. When comparing probiotic-treated females with controls, functional MRI differences were observed in many brain areas, both in basal activity and when participating in an emotional test. These authors hypothesized that the probiotic treatment dampened activity in brain areas regulating emotional and sensory responsiveness. Although the research did not directly address signaling between gut and brain, the pattern of brain activation appeared consistent with vagal communication. Also, Tillisch et al. (2013) and others (Sudo et al., 2004; Hemarajata and Versalovic, 2013) noted that probiotic activity could also cause immune or endocrine changes that alter neural functioning. More research will be necessary to tease apart the various possibilities. Although many aspects of brain functioning are affected by gut probiotic activity, the brain serotonergic system is a likely endpoint for the therapeutic effects reported here. In support of this hypothesis, our mouse OCD-like behaviors were induced by a selective serotonin receptor agonist (RU 24969) and attenuated, in our work and elsewhere (Shanahan et al., 2009, 2011) by fluoxetine, a SRI. It is possible that probiotics attenuated mouse OCDlike symptoms by causing fluoxetine-like changes in brain serotonin signaling. However, in contrast to the widespread effects of systemically administered fluoxetine, probiotics may be more selective, perhaps contributing to their lack of noticeable side effects (Tillisch et al., 2013). Interestingly, OCD and autism spectrum disorder (ASD) are often comorbid, and therefore may share similar etiology. For example, pronounced repetitive behaviors are often associated with both (D’Cruz et al., 2013). Some ASD patients also present GI symptoms, implicating gut microbiota in those cases (Louis, 2012). Similar to effects in OCD patients, administration of 5-HT1B agonists also exacerbates symptoms in ASD patients (Novotny et al., 2004). Thus new treatments that work for OCD may also have efficacy in treating ASD. To our knowledge, our study is the first to use a probiotic to reduce OCD-like symptoms in an animal model of OCD. L. rhamnosus had normalizing effects upon perseverative hyperlocomotion, stereotypic turning, thigmotaxis, and perseverative marble burying, and appeared therapeutically comparable with fluoxetine (a standard treatment for human OCD). On the basis of other work reviewed here, we hypothesize that probiotics may not necessarily be correcting neurological mechanisms that predispose to OCD-like behaviors, but rather may be attenuating the anxiety that enhances their occurrence.

Whether probiotics could serve as a stand-alone or adjunctive treatment for human OCD requires further research.

Acknowledgements This research was supported by undergraduate research grants to P.A.K. and D.N.B. from the Department of Biological Sciences and from the College of Arts and Sciences at Lehigh University, Bethlehem, Pennsylvania. The authors thank Dr Michael Kuchka and Jennifer Colquhoun for assistance in bacterial culturing, and Carly Garrison, Maura Heffernan, and Lynda Asadourian for assistance in behavioral data collection. They also thank Dr Neville Alberto for his advice in the development of this project. Finally, the authors thank three anonymous reviewers and editor Jack Bergman for their helpful comments. Conflicts of interest

There are no conflicts of interest.

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Cariogenic effects of probiotic Lactobacillus rhamnosus GG in a dental biofilm model.

Probiotic Lactobacillus rhamnosus GG therapy and microbiological programming in infantile colic: a randomized, controlled trial.

Towards a better understanding of Lactobacillus rhamnosus GG--host interactions.

Stability of Lactobacillus rhamnosus GG in prebiotic edible films.

Lactobacillus rhamnosus GG supernatant upregulates serotonin transporter expression in intestinal epithelial cells and mice intestinal tissues.

Lactobacillus rhamnosus GG Outcompetes Enterococcus faecium via Mucus-Binding Pili: Evidence for a Novel and Heterospecific Probiotic Mechanism.

Preserving viability of Lactobacillus rhamnosus GG in vitro and in vivo by a new encapsulation system.

Lactobacillus rhamnosus GG protects against non-alcoholic fatty liver disease in mice.

In vivo gut transcriptome responses to Lactobacillus rhamnosus GG and Lactobacillus acidophilus in neonatal gnotobiotic piglets.

Probiotic Lactobacillus rhamnosus inhibits the formation of neutrophil extracellular traps.

Lactobacillus rhamnosus GG Lysate Increases Re-Epithelialization of Keratinocyte Scratch Assays by Promoting Migration.

Lactobacillus rhamnosus GG suspected infection in a newborn with intrauterine growth restriction.

db mice.

Interactions between Lactobacillus rhamnosus GG and oral micro-organisms in an in vitro biofilm model.

Lactobacillus rhamnosus GG-supplemented formula expands butyrate-producing bacterial strains in food allergic infants.

Lactobacillus rhamnosus GG Dosage Affects the Adjuvanticity and Protection Against Rotavirus Diarrhea in Gnotobiotic Pigs.

Lactobacillus rhamnosus GG on rotavirus-induced injury of ileal epithelium in gnotobiotic pigs.

Functional identification of conserved residues involved in Lactobacillus rhamnosus strain GG sortase specificity and pilus biogenesis.

Lactobacillus rhamnosus GG in the middle ear after randomized, double-blind, placebo-controlled oral administration.

Lactobacillus rhamnosus GG and Bifidobacterium longum attenuate lung injury and inflammatory response in experimental sepsis.

Effects of Lactobacillus rhamnosus GG on hepatic and serum lipid profiles in zebrafish exposed to ethanol.

Genome Sequence of the Probiotic Strain Lactobacillus rhamnosus (Formerly Lactobacillus casei) LOCK908.

Effect of Lactobacillus rhamnosus GG Administration on Vancomycin-Resistant Enterococcus Colonization in Adults with Comorbidities.

Probiotic Lactobacillus rhamnosus GG enhanced Th1 cellular immunity but did not affect antibody responses in a human gut microbiota transplanted neonatal gnotobiotic pig model.