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Behavioural Brain Research 302 (2016) 104–114

Contents lists available at ScienceDirect

Behavioural Brain Research

journa l homepage: www.e lsev ier .com/ locate /bbr

esearch report

erotonergic activation of locomotor behavior and posture in one-dayld rats

illary E. Swann, R. Blaine Kempe, Ashley M. Van Orden, Michele R. Brumley ∗

daho State University, Department of Psychology, Pocatello, ID, United States

i g h l i g h t s

Quipazine induced air-stepping, locomotion, and posture in P1 rats.During air-stepping, steps maintained highly anti-phase coordination.Forms of locomotion included pivoting, crawling, and some walking.Advanced posture such as head elevation and locomotor stances were shown.

r t i c l e i n f o

rticle history:eceived 11 July 2015eceived in revised form8 November 2015ccepted 5 January 2016vailable online 12 January 2016

eywords:eonatal ratuipazineocomotionostureteppingnterlimb coordination

a b s t r a c t

The purpose of this study was to determine what dose of quipazine, a serotonergic agonist, facilitatesair-stepping and induces postural control and patterns of locomotion in newborn rats. Subjects in bothexperiments were 1-day-old rat pups. In Experiment 1, pups were restrained and tested for air-steppingin a 35-min test session. Immediately following a 5-min baseline, pups were treated with quipazine (1.0,3.0, or 10.0 mg/kg) or saline (vehicle control), administered intraperitoneally in a 50 �L injection. Bilateralalternating stepping occurred most frequently following treatment with 10.0 mg/kg quipazine, howeverthe percentage of alternating steps, interlimb phase, and step period were very similar between the 3.0and 10.0 mg/kg doses. For interlimb phase, the forelimbs and hindlimbs maintained a near perfect anti-phase pattern of coordination, with step period averaging about 1 s. In Experiment 2, pups were treatedwith 3.0 or 10.0 mg/kg quipazine or saline, and then were placed on a surface (open field, unrestrained).Both doses of quipazine resulted in developmentally advanced postural control and locomotor patterns,including head elevation, postural stances, pivoting, crawling, and a few instances of quadrupedal walk-

ing. The 3.0 mg/kg dose of quipazine was the most effective at evoking sustained locomotion. Betweenthe 2 experiments, behavior exhibited by the rat pup varied based on testing environment, emphasizingthe role that environment and sensory cues exert over motor behavior. Overall, quipazine administeredat a dose of 3.0 mg/kg was highly effective at promoting alternating limb coordination and inducinglocomotor activity in both testing environments.

. Introduction

Mature locomotor behavior involves the ability to alternatelyex and extend the limbs while maintaining postural stability, asell as the ability to traverse various terrains and make changes

n movement direction [1]. The mechanisms supporting locomo-

ion begin developing prenatally in most species, but in altricialnimals, such as rats and humans, a large portion of this develop-ent continues after birth as well. This is largely due to the fact that

∗ Corresponding author at: Department of Psychology, Idaho State University, 921 8th Ave, Stop 8112, United States. Fax: +1 208 282 4832.

E-mail address: brummich@isu.edu (M.R. Brumley).

ttp://dx.doi.org/10.1016/j.bbr.2016.01.006166-4328/Published by Elsevier B.V.

Published by Elsevier B.V.

the ontogeny of locomotion does not involve a single system, butis dependent upon the development of various systems (i.e., sen-sory, motor, postural, neurotransmitter, etc.). This is exemplifiedin the case of the rat [2]. Locomotor-like alternation of the fore-limbs and hindlimbs is expressed as early as gestational day 20 (2days before birth) in the rat [3,4]. While some of the basic motorcoordination mechanisms for locomotion are present prenatally,the newborn must adjust to the demands of a terrestrial environ-ment, such as exhibiting postural control to counteract gravity,to engage in more complex locomotor behavior. During the pre-

natal period, the fetal rat does not have gravitational constraintsplaced on limb movement in utero as it does when attemptinglocomotion in the postnatal environment [5]. This adjustment tolocomotion in a gravitational environment, for an animal with

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elatively weak anti-gravity extensor muscle control [6], may helpo explain why neonatal rats and other altricial newborns exhibitittle spontaneous locomotion at birth.

Over the first two postnatal weeks, rat pups show limited, butradual changes in their locomotor behavior. Immediately afterirth they maintain a flaccid posture with limbs extended awayrom the body [7]. Control of the forelimbs and shoulders of theat begin to mature during the first postnatal week, allowing thenimal to support its weight with the front portion of its body.t the end of the first postnatal week, rat pups begin to exhibitivoting behavior and some crawling. However, it is not until theecond week that the hindlimbs begin to catch up to the forelimbs,ermitting expression of more mature patterns of locomotion,

ncluding more frequent crawling and walking, which graduallyecome more coordinated and adult-like [7]. Changes in locomo-or behavior are accompanied by developmental changes in limb

otoneuron response properties [6], muscle activation patterns8], and descending supraspinal pathways [9].

Given the gradual development of locomotor and posturalehavior, researchers have established the air-stepping paradigmo permit the study of early locomotor development in vivo [10–12].he air-stepping paradigm is a useful model for studying the earlyevelopment of locomotion because it alleviates the gravitationalnd postural constraints of terrestrial locomotion. This procedurenvolves suspending the immature animal in a sling, so it can exhibitocomotor limb activity without the necessity of counteractingravity. Previous studies in our lab, as well as others, have shownhat sustained periods of air-stepping (defined as the limbs mov-ng in a locomotor-like pattern while the animal is suspended offhe floor) can be induced in the neonatal rat by activating the sero-onin or dopamine systems [10–12]. Additionally, air-stepping haseen evoked using olfactory stimuli (i.e., bedding material) [13].hen pups are suspended off the ground and presented with bed-

ing material from the nest, they exhibit air-stepping behavior13]; however, olfactory-induced stepping does not appear to bes sustained as drug-induced stepping.

To evoke air-stepping in rodents with serotonergic stimulation,he serotonin receptor agonist quipazine is often used [4,11,14,15].he effects of quipazine on stepping are blocked by pre-treatmentith a 5-HT2 antagonist, providing evidence that quipazine acts

t 5-HT2 receptors [16,17]. Quipazine-induced stepping has beensed to study the development [4,11], sensory regulation [18,19],nd spinal mechanisms [14,16,15,17,19] of locomotor activity. Aose-response curve conducted in fetal rats in vivo found that.0 mg/kg of quipazine evoked significantly more alternating stepsover 15 times more) compared to saline control subjects [4].owever, since most studies utilize postnatal rat pups in their-stepping paradigm, especially to study mechanisms regulat-ng stepping behavior, it is imperative to examine this issue inostnatal pups. Furthermore, although quipazine evokes steppingehavior, the effect of quipazine dose on interlimb coordinationarameters, such as step period and interlimb phase, has not been

nvestigated. Thus, one purpose of the present study was to con-uct a dose-response curve for quipazine in postnatal rat pups,o assess effective dosage, step period and interlimb phase dur-ng quipazine-induced stepping. This information is important tonow for our understanding of how this air-stepping model relateso coordinated locomotion.

While the air-stepping paradigm provides a model to study earlyevelopment of locomotor behavior, it is important to point outhat air-stepping is not locomotion per se. Locomotion requiresot only movement of the limbs, but also postural control and the

ropulsion of the body through space. Neonatal rats that exhibitir-stepping do not move their entire body through space or acrossn area, but rather only move their limbs while the body remainstationary (i.e., secured to the bar from which they are suspended).

Research 302 (2016) 104–114 105

Crawling in the immature rat has been induced with olfactory stim-uli, such as nest and bedding odor at postnatal day 0 (P0) [13] andamniotic fluid and milk at P1 [20]. But unlike quipazine-inducedstepping, locomotor activity induced by olfactory stimulation isusually very brief in duration. Nonetheless, these experimentsdemonstrate that the newborn rat has the ability to at least showbrief bouts of crawling locomotion.

The duration and types of locomotor and postural patterns (i.e.,crawling, walking locomotion, quadrupedal stance) that quipazineevokes in the freely moving neonatal rat is the second main focusof the present study. Spear and Ristine [21] demonstrated thatquipazine is capable of evoking increased locomotor behavior in P3rats, at a dose of 10.0 mg/kg. Compared to that study, here we testyounger pups, examine locomotion and posture in a larger testingarena, classify several additional locomotor and postural behav-iors, and compare the effect of different quipazine doses on suchbehaviors. Thus the experiments in this study are aimed at assess-ing how closely the quipazine-induced stepping paradigm relatesto actual locomotion and identification of the most effective doseof quipazine for evoking locomotor behavior in neonatal rats.

2. General methods

2.1. Subjects

Subjects were Sprague-Dawley rats bred in the Animal CareFacility at Idaho State University. Subjects remained housed in thehome cage with the dam until testing. Testing occurred on P1 (24 hafter birth). Animals were examined prior to testing to ensure thatthey had fed recently as indicated by the presence of a milk band onthe abdomen, and were in overall good health (e.g., pink in color).A total of 56 P1 rat pups were used as subjects in the two exper-iments. In order to avoid litter effects [22], no more than one pupper litter was assigned to each group. Animal care and use were inaccordance with NIH Guidelines [23] and the Idaho State UniversityAnimal Care and Use Committee.

2.2. Behavioral testing

On day of testing, pups were individually tested inside an incu-bator that maintained humidity (∼40%) and ambient temperature(at 35 ◦C). They were manually voided for up to 20 s or until uri-nation/defecation, by gently stroking the perineum with a smallpaintbrush. Pups were then placed inside the incubator for 30 minprior to testing to allow for acclimation to testing conditions. Fol-lowing acclimation, pups received an intraperitoneal (IP) injectionof quipazine or saline.

2.3. Pharmacology

Quipazine maleate, a serotonergic receptor agonist(Sigma–Aldrich, St. Louis, MO) was prepared in doses of 1.0 mg/kg,3.0 mg/kg, or 10.0 mg/kg. Doses administered were based on aprevious study in fetal rats [4], as well as previous studies withnewborn rats that used 3.0 mg/kg quipazine to evoke alternat-ing stepping [10,11,18,19]. Pups in the control group for bothexperiments received saline (vehicle control). Drugs were admin-istered through a 0.05 mL IP injection with a 30-gauge needle. Theresearcher was blind to drug condition during administration.

3. Experiment 1: effect of quipazine dose on interlimb

coordination and step period

Although quipazine is often used to evoke locomotor-like step-ping behavior in newborn rats in vivo, step coordination during

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uipazine-induced stepping has not been characterized. The pur-ose of this experiment was to examine the step coordinationarameters of interlimb phase and step period, in addition to step

requency, at different doses of quipazine administration in 1-day-ld rats. In accord with a dose-response curve conducted withetuses [4], we hypothesized that the 3.0 mg/kg quipazine dose

ould evoke more alternating steps than saline or other doses ofuipazine.

.1. Methods

.1.1. DesignA total of 32 P1 rats (16 males, 16 females) were used as subjects

n Experiment 1. Subjects were assigned to one of four drug con-itions: 1.0 mg/kg, 3.0 mg/kg, or 10.0 mg/kg quipazine, or saline.ollowing a 30-min acclimation period to incubator conditions,ubjects were secured to a soft, rubber-covered horizontal bar inhe prone posture, using a harness. The harness allowed the fore-imbs and hindlimbs to hang and move freely meanwhile keepinghe body secured to the bar [18]. Once the subject was securelyuspended from the bar, a 5-min baseline was recorded. Immedi-tely following baseline, pups received an IP injection of quipaziner saline and behavior was recorded for 30 min.

The test session comprised a 5-min baseline and 30-min periodost-injection. The length of the test session was determined basedn a previous study [18] which found a significant increase intepping behavior within 5 min following quipazine administra-ion and that lasted for at least 45 min. Several studies have thustilized only a 30-min test session post-injection [11,19], as thateems sufficient to capture and analyze sustained stepping behav-or. The session was recorded from a microcamera located directlynderneath the pup and able to capture all limb movements. Theicrocamera was connected to a DVR recording unit outside the

ncubator. SMPTE longitudinal time-code was impressed on videoecordings throughout the test session.

.1.2. Behavioral scoringThe 35-min test session was recorded and then scored during

VD playback at normal or reduced speed. Behavior was scoredn two scoring passes: one for scoring forelimb activity and oneor scoring hindlimb activity. Events were entered into an eventecorder program (JWatcher Version 1.0) [24], which records theategory of behavior and time of entry (±0.01 s). The experimenteras blind to subject’s group association. Intra- and interrater reli-

bility for scoring were >90%.

.1.2.1. Step frequency. An alternating step is an alternating pat-ern of consecutive flexion and extension of homologous limbs17]. Thus in this study alternating steps are a bilateral movementattern, counted only when both limbs (both forelimbs or bothindlimbs) show alternating stepping movements. For this experi-ent, all other limb activity that did not fall into the “step” categoryas scored as a non-stepping limb movement. Separate scoring

asses were conducted for the forelimbs and hindlimbs, indicat-ng each incidence of alternating stepping and (non-stepping) limbctivity. Additionally, the percentage of alternating steps as a func-ion of total limb movements was determined.

.1.2.2. Interlimb phase and step period. To calculate interlimbhase and step period, the first sequence of 3–5 alternating steps (asefined above) that occurred within 2-s of each other was selected

er 5-min time bin for each subject. Interlimb phase and step periodere then determined for each step in each sequence, by using

he right limb retraction onset as a reference: this was the pointhen the right limb began to flex as the left limb began to extend.

Research 302 (2016) 104–114

Interlimb phase provides a measurement of interlimb coordina-tion. When limbs are moving in perfect synchrony, interlimb phaseis 1.0; when limbs are moving in a perfect anti-phase or alternat-ing pattern, interlimb coordination is 0.50. Step period is the timethat is required for a complete alternating step cycle to occur. Thisanalysis was restricted to the 30-min period post-injection only, asvery few alternating steps occurred during baseline.

3.1.3. Statistical analysisAnalyses were conducted separately for forelimbs and

hindlimbs. Behavior was summarized into 5-min time binsacross the test session to examine time-dependent changes inquipazine-induced stepping activity, using repeated measuresANOVA (with time as the repeated measure). Independent vari-ables were quipazine dose and time. Dependent variables werealternating step frequency, percentage of alternating steps, inter-limb phase, and step period. Tukey’s post-hoc comparisons ofmeans were conducted following significant main effects and/orinteractions. IBM SPSS Statistical Software (Version 22) was usedfor analysis and a 5% significance level was adopted for all tests.

3.2. Results

3.2.1. Forelimb steppingA two-way repeated measures ANOVA (4 doses × 7 time bins)

revealed a main effect of dose (F(3,28) = 41.64, p < .001), a maineffect of time (F(6,168) = 37.2, p < .001), and an interaction of thetwo factors (F(18,168) = 10.04, p < .001) on alternating forelimb stepfrequency. As can be seen in Fig. 1(A), all 3 doses of quipazineevoked significantly more alternating forelimb steps compared tosaline at all time points, except for baseline (T0) and the first 5-minfollowing baseline (T5) for the 1.0 mg/kg dose. Additionally, the 3.0and 10.0 mg/kg doses of quipazine evoked significantly more fore-limb steps than the 1.0 mg/kg dose of quipazine, following injection.

For percentage of alternating forelimb steps, a two-wayrepeated measures ANOVA (4 doses × 7 time bins) revealed amain effect of dose (F(3,28) = 49.12, p < .001), a main effect of time(F(6,168) = 67.68, p < .001), and an interaction between dose andtime (F(18,168) = 7.84, p < .001). As shown in Fig. 1(B), all doses ofquipazine evoked a significantly higher percentage of alternatingforelimb steps compared to saline at all time points, except at T0and at T5 for the 1.0 mg/kg dose.

Because the 3.0 and 10.0 mg/kg doses of quipazine evokedthe highest frequency and most consistent forelimb stepping, weexamined interlimb phase and step period at these doses. Analy-ses were conducted during the 30-min period following injection.A two-way repeated measures ANOVA (2 doses × 6 time bins)revealed no significant difference between 3.0 and 10.0 mg/kgdoses of quipazine for forelimb interlimb phase, with limbs averag-ing a phase relationship of 0.48 ± 0.09 (Fig. 1C). This indicates thatthe forelimbs were in near perfect alternation. For step period, therewas no significant difference across quipazine doses for forelimbstep period. Additionally, forelimb step period remained relativelyconstant across the test session (Fig. 1D), with an average forelimbstep period of 0.92 ± 0.28 s.

3.2.2. Hindlimb steppingFor frequency of alternating hindlimb steps, a two-way repeated

measures ANOVA revealed a main effect of dose (F(3,28) = 30.23,p < .001), a main effect of time (F(6,168) = 43.16, p < .001), and aninteraction between dose and time (F(18,168) = 10.31, p < .001). Asshown in Fig. 2(A), all doses of quipazine significantly increased

the frequency of alternating hindlimb steps compared to saline.As with the forelimbs, the 3.0 and 10.0 mg/kg doses of quipazineevoked the most steps. For percentage of alternating hindlimbsteps, a two-way repeated measures ANOVA revealed a main

H.E. Swann et al. / Behavioural Brain Research 302 (2016) 104–114 107

Fig. 1. Alternating forelimb air-stepping following treatment with quipazine. (A) Frequency of alternating forelimb steps. T0 represents the baseline period, while T5r forelit 10.0 mc

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epresents the first time bin following drug injection. (B) Percentage of alternatinghe 3.0 and 10.0 mg/kg doses of quipazine. (D) Forelimb step period at the 3.0 and

onducted following baseline. Points indicate means; vertical lines depict SEM.

ffect of dose (F(3,28) = 23.18, p < .001), a main effect of timeF(6,168) = 65.04, p < .001), and an interaction between these twoactors (F(18,168) = 6.04, p < .001). All doses of quipazine evokedignificantly higher percentages of hindlimb steps compared toaline (Fig. 2B).

For hindlimb interlimb phase and step period, analyses wereestricted to the 3.0 mg/kg and 10.0 mg/kg doses of quipazine. Awo-way repeated measures ANOVA revealed no significant differ-nce between the two doses of quipazine in regards to interlimbhase. As can be seen in Fig. 2(C), interlimb phase remained fairlyonsistent across the test session with a mean of 0.51 ± 0.08. Therelso was not a significant difference between doses of quipazinen hindlimb step period. However, a two-way repeated measuresNOVA did indicate time-dependent changes in hindlimb steperiod (F(5,60) = 3.45, p < .05). As shown in Fig. 2(D), hindlimb steperiod initially started just below 1 s (at T5), but then increased tover 1 s consistently from T15–T30.

The results from Experiment 1 indicate that quipazine evokesigh frequencies of alternating stepping in both the forelimbsnd hindlimbs. Additionally, quipazine induces highly coordinatedilateral stepping behavior in both limb pairs, as the limbs main-

mb steps as a function of all forelimb movements. (C) Forelimb interlimb phase atg/kg doses of quipazine. Note: For interlimb phase and step period, analyses only

tained a pattern of anti-phase interlimb coordination throughoutthe test period following treatment with quipazine. Previous stud-ies have utilized a 3.0 mg/kg dose of quipazine to evoke alternatinglimb activity in fetal and postnatal rats [4,11]. However, it wasunclear if 3.0 mg/kg dose of quipazine was the most effective doseof quipazine to evoke highly anti-phase coordination in P1 ratpups. Findings from this experiment indicate that a dose of 3.0or 10.0 mg/kg quipazine is effective at evoking highly coordinatedalternating stepping behavior in newborn rats.

4. Experiment 2: quipazine administration promotesadvanced locomotion and posture

To determine if quipazine facilitates posture and locomotion inP1 rats, subjects were treated with the most effective quipazinedoses (3.0 or 10.0 mg/kg) from the first experiment and placed

in an open-field (unrestrained) where their postural stability andlocomotor patterns were measured. Whether or not quipazinefacilitates locomotor behavior in the freely moving rat is importantto know, as it would help to validate the use of quipazine-induced

108 H.E. Swann et al. / Behavioural Brain Research 302 (2016) 104–114

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ig. 2. Alternating hindlimb air-stepping following treatment with quipazine. (A) Function of all hindlimb movements. (C) Hindlimb interlimb phase at the 3.0 and 10uipazine. Points indicate means; vertical lines depict SEM.

tepping as a model of locomotion in the perinatal rat and deter-ine if postural control, in addition to limb activity, is promoted by

erotonergic stimulation as well. We hypothesized that the 3.0 and0.0 mg/kg doses of quipazine would evoke comparable rates of

ocomotor and postural behavior given their similar effects on air-tepping, but more locomotor and postural behavior compared toaline-treated subjects. Because unrestrained locomotor behaviorollowing quipazine administration has not been examined pre-iously, the test session was lengthened to 45-min. This was inase patterns of locomotion take longer to activate compared to air-tepping (i.e., perhaps due to having to maintain postural controln order to express locomotion).

.1. Methods

.1.1. Design

A total of 24 P1 rats (12 male, 12 female) were used as subjects

n this experiment. On the day of testing, pups were marked on theentrum using a non-toxic black marker for tracking purposes. Fol-owing a 30-min acclimation period, pups received an IP injection

ncy of alternating hindlimb steps. (B) Percentage of alternating hindlimb steps as a/kg doses of quipazine. (D) Hindlimb step period at the 3.0 and 10.0 mg/kg doses of

of 3.0 or 10.0 mg/kg quipazine, or saline. After injection, pups wereplaced unrestrained on a 10 × 12 in clear, Plexiglas surface markedwith gridlines. A mirror was placed at a 45◦ angle underneath thePlexiglas surface and a Sanyo Xacti FH1 video camera (model no.VPC-FH1, 60 fps) was positioned outside the incubator, which cap-tured both a lateral and ventral view of the subject. Test sessionsbegan immediately following drug injection and continued for aperiod of 45 min. The test session was recorded onto an internal SDcard and then copied to DVD at a later time.

4.1.2. Behavioral scoringThe 45-min test session was scored during video playback at

normal or reduced speed. Locomotor behavior was classified into 3categories (walking, crawling, and pivoting), using the classificationof Altman and Sudarshan [7]. Walking was defined as a propulsivemovement that involved all four limbs with the belly off the sur-

face. Crawling was a propulsive movement, which actively involvedthe forelimbs with the belly on the surface. Pivoting was a propul-sive movement where the pelvis remained on the surface while theforelimbs propelled the pup in a circular path. Pivoting typically is

Brain Research 302 (2016) 104–114 109

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H.E. Swann et al. / Behavioural

rst expressed spontaneously at P4-5, crawling is shown as earlys P8, and walking emerges at P12–13 in rats [7].

Postural behavior was classified into 3 categories: head eleva-ion, walking stance, and crawling stance. These postural behaviorsere scored when they happened independently of locomotor

ehavior. Head elevation was any elevation of the head indepen-ent of all other behavior. Walking stance was a postural stanceith both of the forelimbs and hindlimbs extended and the belly off

he floor, without any propulsive movement. Crawling stance was postural stance with forelimbs extended without any propulsiveovement.

Non-locomotor activity was classified into 3 categories: supina-ion, pronation, and limb activity. Supination was used to indicatehen pups were in the supine position, i.e., rolling, and alter-

atively, pronation was used when the animals were in a proneosition, i.e., righting. Limb activity was defined as any limbovement that was not accompanied by a forward or backward

rogression. Behavioral events were scored using JWatcher. Thecorer was blind to drug condition during behavioral scoring.ntrarater reliability was >90%.

.1.3. Crawling distance measurementWe examined the total distance and mean distance travelled

uring each bout of crawling. Using the mark on the ventrum ofhe pup, crawling paths were traced and then measured to deter-

ine distance travelled. Path tracing was conducted on a computercreen, with the gridlines on the bottom of the Plexiglas surface pro-iding measurement scaling. This analysis could not be conductedor pivoting: due to the nature of the pivoting movement, pivotingaths could not be reliably measured using the forelimbs or head as

tracking point. Since the forelimbs are moved to push the animaln a circular movement, the paws are not a consistent and reliableracking point, nor does the head move in a consistent manner withhe body. This analysis was not conducted for walking, as only veryew bouts of walking were shown. Therefore, these analyses wereimited to crawling.

.1.4. Data analysisA one-way ANOVA was conducted for each category of locomo-

ion, posture and non-locomotor activity for frequency and totaluration of behavior over the entire 45-min test session. Tukey’sost-hoc tests were used to compare group differences. Statisticalests were performed using IBM SPSS Statistical Software (Version2), and a 5% significance level was adopted for all tests.

.2. Results

.2.1. Frequency of locomotor and postural behaviorOne-way ANOVAs revealed an effect of quipazine dose on piv-

ting frequency (F(2,21) = 12.30, p < .001) and crawling frequencyF(2,21) = 19.08, p < .001). Subjects treated with 3.0 or 10.0 mg/kguipazine showed significantly more pivoting and crawling com-ared to saline controls (see Fig. 3A). There was no difference inivoting frequency between subjects treated with 3.0 or 10.0 mg/kguipazine; however the 10.0 mg/kg dose induced significantlyore instances of crawling than the 3.0 mg/kg dose or saline, and

he 3.0 mg/kg dose induced significantly more instances of crawl-ng than saline. There was no effect of dose on walking, as walkingery seldom occurred (Fig. 3A). Note that walking was not exhib-ted by saline-treated subjects, but only occurred a few times inuipazine-treated subjects.

One-way ANOVAs indicated no effect of dose on the frequency of

rawling stance or head elevation, but there was an effect of dosen walking stance frequency (F(2,21) = 3.87, p < .05). As shown inig. 3(B), subjects treated with 3.0 mg/kg quipazine showed sig-ificantly higher frequencies of walking stance than saline-treated

and limb activity. Bars show means; vertical lines depict SEM.

110 H.E. Swann et al. / Behavioural Brain

Fig. 4. Total duration of locomotor and postural behavior during the 45-min testppw

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eriod following administration of quipazine, in the open field. (A) Total duration ofivoting, crawling, and walking locomotion. (B) Total duration of crawling stance,alking stance, and head elevation. Bars show means; vertical lines depict SEM.

ubjects. There was no difference between 3.0 and 10.0 mg/kg ofuipazine or saline and 10.0 mg/kg quipazine.

There was an effect of dose on frequency of supinationF(2,21) = 19.76, p < .001), pronation (F(2,21) = 19.2, p < .001), andimb activity (F(2,21) = 8.78, p < .05). As shown in Fig. 3(C), sub-ects treated with 10.0 mg/kg quipazine showed significantly morenstances of supination and pronation than subjects in other groups.here was no difference between saline and 3.0 mg/kg quipazine.dditionally, subjects treated with 3.0 or 10.0 mg/kg quipazinehowed significantly more limb activity than saline-treated sub-ects. There was no difference between 3.0 and 10.0 mg/kguipazine (Fig. 3C).

.2.2. Duration of locomotor and postural behaviorOne-way ANOVAs indicated an effect of dose on duration of

ivoting (F(2,21) = 16.13, p < .001) and crawling (F(2,21) = 10.90, < .001), but not on walking (walking very rarely occurred).ubjects treated with the 3.0 mg/kg dose of quipazine showed

ignificantly longer durations of pivoting than subjects treatedith the 10.0 mg/kg dose or saline; also, subjects treated with the

0.0 mg/kg dose showed significantly longer durations of pivotingompared to saline controls (Fig. 4A). For crawling duration, sub-

Research 302 (2016) 104–114

jects treated with 3.0 or 10.0 mg/kg quipazine showed significantlylonger durations of crawling compared to saline controls (Fig. 4A).

A series of one-way ANOVAs revealed no effect of dose on dura-tions of crawling or walking stance; however, there was effect ofdose on head elevation duration (F(2,21) = 4.12, p < .05; Fig. 4B).Subjects treated with 3.0 mg/kg quipazine demonstrated signif-icantly longer durations of head elevation than saline controlsor subjects treated with 10.0 mg/kg quipazine. There was no dif-ference between subjects treated with 10.0 mg/kg quipazine andsaline controls.

4.2.3. Locomotor patterns over time for quipazine-treated pupsIn addition to total duration, we also looked at the expression of

locomotor patterns over time for quipazine-treated pups. For thisanalysis, durations of different locomotor behaviors were summa-rized into 3-min time bins across the 45-min test session (for a totalof 15 3-min time bins). Because we found such high frequenciesand total durations of pivoting and crawling in pups treated withquipazine, we examined here if developmentally earlier locomotorpatterns (pivoting) would occur earlier during the test session thana developmentally more mature locomotor pattern (crawling) thatalso requires more postural control. Saline-treated controls did notshow enough pivoting and crawling to warrant such an analysis.

Two-way repeated measures ANOVAs (2 doses × 15 time bins)were conducted separately for pivoting and crawling. Therewas no effect of time on pivoting, nor an interaction betweendose and time. There was a main effect of time on crawling(F(14,196) = 4.47, p < .001) and an interaction between time anddose (F(14, 196) = 3.73, p < .001). As can be seen in Fig. 5(A), crawlingduration was initially higher for subjects treated with 10.0 mg/kgquipazine. Next, dependent t-tests compared durations of pivot-ing and crawling at each 3-min time bin. This was done separatelyfor the two doses of quipazine. For subjects treated with 3.0 mg/kgquipazine, significantly more crawling than pivoting occurred dur-ing the first three minutes of the test session, t(7) = −2.90, p < .02(Fig. 5B). However, there were no effects found in the othertime bins. As shown in Fig. 5(C), subjects treated with 10.0 mg/kgquipazine demonstrated higher durations of crawling, not pivoting,earlier in the test session (see T6–T24 on Fig. 5C). Taken together,these analyses show there was no strong evidence for pivoting orcrawling happening earlier or later during the test session for pupstreated with 3.0 or 10.0 mg/kg quipazine.

4.2.4. Crawling distanceThis analysis was limited to subjects treated with 3.0 mg/kg

quipazine or saline. We found that subjects treated with 10.0 mg/kgquipazine did not exhibit sustained crawling paths that could betraced using the present methodology. While subjects treated withthe 10.0 mg/kg dose showed increased crawling over the 45-mintest session, we did not find that they showed longer durations ofcrawling compared to the 3.0 mg/kg dose; additionally, we foundthat their individual crawling bouts were frequently interruptedwith supination and pronation behavior (i.e., rolling). Thus the mostsustained crawling was observed in subjects treated with 3.0 mg/kgquipazine, and thus crawling distance was measured in these sub-jects to examine quipazine-induced crawling.

As shown in Fig. 6(A), quipazine-treated pups covered asignificantly greater total distance by crawling compared to saline-treated subjects, t(14) = −3.83, p < .01. However, as shown inFig. 6(B), there was not a significant effect of drug on mean distanceper bout of crawling. Fig. 6(C) and (D) shows sample crawling pathsfor two individual pups treated with 3.0 mg/kg quipazine. Often

times pups would show a brief bout of crawling, roll (alternatebetween supination and pronation) for a second or two, and thenbegin showing another locomotor behavior. Taken together withthe above results, this suggests that quipazine mainly increased the

H.E. Swann et al. / Behavioural Brain

Fig. 5. Locomotor patterns across time for quipazine-treated pups in the open field.(A) Crawling duration for pups treated with 3.0 or 10.0 mg/kg quipazine. Time isexpressed in 3-min bins on the x-axis. (B) Expression of pivoting and crawling forppS

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ups treated with 3.0 mg/kg quipazine. (C) Expression of pivoting and crawling forups treated with 10.0 mg/kg quipazine. Points show means; vertical lines depictEM.

requency of crawling bouts, but not the length/distance covered byach individual crawling bout.

. Discussion

The serotonergic agonist quipazine evoked highly coordinatedlternating stepping in P1 rats at the 3.0 and 10.0 mg/kg doses.

t each of theses doses, quipazine-treated subjects demonstratedigh frequencies of alternating steps, highly consistent anti-phase

nterlimb coordination, as well as maintained a fairly constant steperiod, for both the forelimbs and hindlimbs. Though it is interest-

Research 302 (2016) 104–114 111

ing to point out that the forelimbs maintained a nearly constantstep period across the test session, whereas the hindlimbs showeda slight increase (longer duration) of step period. This may be dueto weaker hindlimb muscles [25] being unable to sustain such arapid step period, regardless of quipazine administration. However,this increase in step period did not affect the stepping coordination(interlimb phase). Overall, we found that while subjects treatedwith 10.0 mg/kg quipazine demonstrated slightly elevated fre-quencies of alternating stepping, that there was not a concomitantincrease in other stepping parameters. There were no differencesbetween the 3.0 and 10.0 mg/kg doses of quipazine for percent-age of alternating steps, interlimb phase, or step period. Both doseseffectively evoked highly coordinated, alternating stepping behav-ior. Given this, we suggest that 3.0 mg/kg quipazine is an effectivemeans of evoking air-stepping behavior.

Quipazine also facilitated locomotion and postural stability inthe freely moving newborn rat. Quipazine-treated subjects demon-strated developmentally advanced locomotor patterns, includingpivoting (typically seen at P4) and crawling (typically seen at P8)[7]. Additionally, quipazine evoked increased postural behavior,such as walking stance and head elevation. Although crawlingstance alone did not increase following quipazine administra-tion, this posture is involved in supporting crawling behavior andtherefore also increased in this manner. Non-locomotor behav-ior, including supination (rolling), pronation (righting), and limbactivity, also increased following quipazine administration. Thus,quipazine increased the frequency and duration of locomotor andpostural behavior 1–2 weeks earlier than usual, and did so over arelatively sustained course of the 45-min test session (see Fig. 5).To our knowledge, this is the earliest demonstration of sustainedlocomotion (not just alternating stepping activity) in the newbornrat. These findings build upon the Spear and Ristine [21] paperthat found an increase in forward locomotion following quipazineadministration in three-day-old rats.

Our finding that activation of the serotonin system facilitatesposture and locomotion in newborn rats is logically consistent withstudies that have examined serotonin depletion during early devel-opment. Administration of p-chlorophenylalaine (PCPA) blocksserotonin synthesis, and when administered during the early post-natal period has been shown to significantly retard locomotor andpostural development in rats. Following PCPA treatment duringthe first two postnatal weeks, rat pups exhibited a lack of pos-tural stability and control [26]. Rat pups also showed decreasedcoordination of forelimb and hindlimb movements [27]. In thecurrent study, activation of the serotonin system with quipazinefacilitated a variety of locomotor and postural behaviors. How-ever, postural stability was largely seen in the context of locomotorbehavior than as independent postural stances. In fact, we foundthat quipazine-treated subjects did not cover greater distances inindividual crawling bouts compared to saline-treated controls, butrather showed an increase in the total distance covered as a functionof increased crawling bouts. This suggests that pups may still lackthe muscle strength necessary to exhibit continued or sustainedpostural stability. Previous research has shown that high frequencyfiring in the extensor muscles, typical of adult locomotion, is notshown until P15 [8]. Thus, relatively weak muscles are likely tocontribute to difficulty in exhibiting sustained locomotor behaviorprior to P15. It is also interesting to note that serotonergic receptorsmight differentially affect motor mechanisms, possibly contribut-ing the findings reported here. It has been suggested that 5-HT2agonists influence extensor activity, while 5-HT7 and 5-HT1A recep-tor agonists influence hindlimb-walking alternation [28]. Thus, it

could also be possible that a lack of sustained locomotor behav-ior exhibited by quipazine-treated subjects is a result of promotedextensor activity, but not left-right alternation as suggested bySlawinska et al. [28].

112 H.E. Swann et al. / Behavioural Brain Research 302 (2016) 104–114

Fig. 6. Crawling distance and trajectories for pups treated with 3.0 mg/kg quipazine, in the open field. (A) Total distance travelled by crawling for quipazine- and saline-treateds treatef arrowp 1 indi

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ubjects. (B) Mean distance per bout of crawling exhibited by quipazine- and saline-or two quipazine-treated subjects. Starting point is indicated by a solid black dot;ronation, pivoting, etc.) that account for breaks between crawling paths. Number

Evoking locomotor and postural behavior in immature animalsighlights the importance of testing environment and sensory reg-lation of motor behavior during early development. For example,

t has been shown that facial wiping in rat pups [29] and steppingn human infants [30] is facilitated or hindered by the testing envi-onment. In the current study, we found that pups differentiallyespond to quipazine based on the testing environment. Whenuspended in the air, quipazine-treated pups showed alternat-ng stepping behavior without drastic differences in coordinationnd frequency across the 3.0 and 10.0 mg/kg doses of quipazine.owever, when placed on a Plexiglas surface, pups demonstratedivoting and crawling, as well as other postural stances andon-locomotor behavior, and importantly, we find differences

n behavior exhibited between the doses of quipazine. Subjectsreated with 10.0 mg/kg quipazine demonstrated increased fre-uency, but not duration, of crawling. Rolling and righting behavior

nterrupted these bouts of crawling, whereas subjects treated with.0 mg/kg quipazine demonstrated crawling that was not inter-upted as frequently by supination and pronation, thus, resultingn longer individual bouts of crawling behavior.

Based on this, we conclude that the most effective dose atvoking alternating stepping, locomotion, and postural control forewborn rats is 3.0 mg/kg quipazine. Furthermore, we have con-dence that the air-stepping paradigm is a valid methodologicalpproach for examining the development and function of loco-otor circuitry in newborn rats. While quipazine induces actual

ocomotor and postural behavior, these behaviors are not expressedt a consistent rate as found with air-stepping. Therefore, their-stepping paradigm provides a model to examine sustained loco-

otor activity that is limited on a flat surface or in the freely moving

nimal.Pups tested on the Plexiglas surface had access to cutaneous

timulation from the testing surface and also likely experienced

d subjects. Bars indicate means; vertical lines depict SEM. (C and D) Crawling paths indicates stop point. Subjects exhibited a number of other behaviors (supination,

cates first bout of crawling, number 2 indicates second bout of crawling, and so on.

gravitational limb loading and differential vestibular and propri-oceptive stimulation during ongoing movement. While previousresearch has found that pups exposed to a stiff (Plexiglas) sub-strate modulated their stepping behavior to apparently avoidcontact with the substrate [18], it seems unlikely that the differ-ence seen in behavior across quipazine doses stems from exposureto the Plexiglas surface, given that all subjects were exposed tothe same substrate. In fact, research has found that limb loadingand weight-bearing postural stances against resistance surfacesincreases extensor muscle activity [31,32]. It has been suggestedthat quipazine activates the output stage of locomotor circuitryby directly activating motoneurons and interneurons of centralpattern generators, thus leading to improved extensor activity[28]. Given that postural stances require limb extension, we mightexpect to see that quipazine increases postural stance and sustainedcrawling. While it is unclear exactly what is causing the differencesacross doses of quipazine, observations seem to indicate that the10.0 mg/kg dose does produce elevated levels of activity. Althoughthese levels are not noticeable in the air-stepping paradigm, signifi-cant differences do emerge in a testing environment that introducescutaneous sensory stimulation. Interestingly, Ichiyama et al. [33]found quipazine dose-dependent changes in coordination in adultspinal rats. Higher doses of quipazine resulted in a decrease incoordinated movement, as well as the number of plantar steps.These animals displayed overly extended and flexed hindlimbs thatimpeded stepping behavior [33]. It seems likely that higher dosesof quipazine overly excite the motor system creating interferencewith the animal’s ability to correctly adjust limb posture, thus pre-venting the coordinated movement and plantar stepping necessary

to sustain locomotor and postural behavior. Indeed, these findingswould appear to map closely to quipazine effects in spinal adultanimals, in that higher doses of quipazine can result in detrimentaleffects on locomotor behavior [34]. Furtermore, it has been sug-

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ested that afferent feedback plays a significant role in the effectsf quipazine on stepping behavior, given that step quality follow-

ng quipazine treatment is dependent upon testing posture (e.g.,pright or horizontal posture) [34].

It is important to note that spinal adult rodents often respondo lower doses of quipazine compared to the doses utilized withostnatal rat pups. We argue that this is largely due to develop-ental differences in 5-HT’s effects on neural populations involved

n locomotion. Specifically, it has been demonstrated that therere important age-dependent differences in spinal V2a interneu-ons that contribute to locomotor behavior [35]. Husch et al. [35]emonstrated that compared to younger animals, adult mice dis-lay increased excitation of V2a interneurons, resulting in changes

n input resistance, as well as shifts in action potentials (narrowerPs in adult mice) and increased 5-HT bistability, which is critical

or producing rhythmic activity within the locomotor CPG [35]. Theulmination of these differences suggests that as the animal ages, aower threshold of activation is required for CPG involvement. Thust would seem to suggest that the application of quipazine wouldifferentially affect these interneurons, resulting in a decrease inhe dose needed to evoke coordinated locomotor behavior in adultnimals compared to neonates. Another important component ofhe 5-HT system is that 5-HT2A receptors seem to facilitate chlo-ide homeostasis following spinal cord injury [36]. Following spinalnjury, there is a disruption in chloride homeostasis, resulting inecreased inhibition. Thus, less stimulation is needed to result inctivity within neural populations. It has been demonstrated thatpplication of a 5-HT2A agonist will result in a shift back to pre-njury levels of inhibition [36], which suggests that dose differencesmong ages do not necessarily arise from disruption in chlorideomeostasis. Additionally, following spinal injury there is an up-egulation of 5-HT receptors in the spinal cord [37,38]. Perhapsp-regulation of 5-HT receptors and restoration of chloride homeo-tasis interact and result in increased sensitivity to serotonergicctivation in spinal adult animals, thus contributing to differencesn doses across age groups and preparations.

Finally, the findings and paradigm used here recognize themportance of examining the plasticity of the motor systemrom a developmental perspective. Establishing a rodent modelo study early motor development allows researchers to, in aontrolled environment, manipulate individual components ofeveloping motor systems, such as neurotransmitter systems, neu-al pathways, and even environmental context (i.e., gravitationalonstraints, sensory stimuli) through systematic and targetedxperimental designs. This approach then has implications for theevelopment of therapeutic interventions for non-typical develop-

ng infants, in that it advances our understanding and knowledgef what developmental processes are occurring and how thoserocesses can be influenced by physiology, sensory feedback, andnvironmental experiences. For example, Teulier et al. [39] demon-trated that contextual manipulation, primarily through sensoryxperiences, influences human infants’ motor behavior. Whennfants are given practice with weight-supported treadmill step-ing, there is improvement in later outcomes [39]. The researchf Thelen and colleagues [30] demonstrated that the disappearingewborn stepping reflex could be evoked when infants were sub-erged in water, emphasizing the critical role that environment

nd gravity exert on locomotor behavior, closely resembling dif-erences between the air-stepping and open field paradigm usedere to examine locomotor behavior. However, Barbu-Roth et al.40] found that gravitational constraints are not the only influencen newborn stepping in human infants, but in fact, this response

ould be influenced and manipulated by visual field stimuli. Over-ll, utilizing basic research, as well as evidence-based pediatricesearch, allows us to examine and manipulate systems involved

[

[

Research 302 (2016) 104–114 113

with coordination and locomotion [10], influencing approaches tointerventions in pediatric populations [39].

6. Conclusions

The findings reported here show that quipazine induces highlycoordinated motor coordination in the perinatal rat, includingadvanced locomotor and postural patterns of behavior at a muchearlier developmental age than seen during typical development.Quipazine-treated subjects demonstrated high frequencies of alter-nating steps, highly consistent anti-phase interlimb coordination,as well as maintained a fairly constant step period, in both theforelimbs and hindlimbs. The behavior exhibited by the rat pupvaried based on testing environment (restrained and suspendedduring air-stepping, or freely moving on a Plexiglas surface),highlighting the role that environment and sensory cues exertover motor behavior. Overall, quipazine administered at a doseof 3.0 mg/kg was highly effective at inducing locomotor activityin both testing environments. The findings here lay importantgroundwork for future studies manipulating serotonergic andlocomotor mechanisms and help with our understanding of thecoordination of quipazine-induced locomotor behavior, includingalternating stepping. Future studies could involve combining dif-ferent methodological approaches for studying locomotor behaviorsuch as examining how activation or blockade of the seroto-nergic system influences olfactory-induced locomotion, or howcoordination parameters are altered following spinal injury andpharmacological stimulation.

Acknowledgements

This study was supported by NIH grant 1R15HD062980-01 toMRB, NIH grant P20GM103408 as part of the INBRE program, andHSSRC grant HR11-6 from Idaho State University to HES.

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