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Behavioural Brain Research 246 (2013) 168– 178

Contents lists available at SciVerse ScienceDirect

Behavioural Brain Research

j ourna l h o mepa ge: www.elsev ier .com/ locate /bbr

esearch report

ead movement: A novel serotonin-sensitive behavioral endpoint forail suspension test analysis

mber Lockridgea, Brett Newlanda, Spencer Printena, Gabriel E. Romeroa,i-Lian Yuana,b,∗

Department of Neuroscience, University of Minnesota, 6-145 Jackson Hall, 321 Church St. SE, Minneapolis, MN 55455, USAGraduate Program in Neuroscience, University of Minnesota, 6-145 Jackson Hall, 321 Church St. SE, Minneapolis, MN 55455, USA

i g h l i g h t s

Head movement is a novel behavioral endpoint in the FST and TST.Reproducible in 129 and C57 mouse strains.Inversely correlated to swimming and selectively enhanced by SSRIs.Weighted TST analysis differentiates 5-HT and norepinephrine responsive behaviors.5-HT2A and 5-HT2C postsynaptic receptor activation diminish TST head movement.

r t i c l e i n f o

rticle history:eceived 15 November 2012eceived in revised form 8 February 2013ccepted 25 February 2013vailable online 13 March 2013

eywords:STST

a b s t r a c t

The tail suspension test (TST) as an antidepressant and depression-related behavior screen, has manyadvantages over the forced swim test (FST) in terms of procedural simplicity and consistent SSRI response.However, the FST has traditionally offered more specific neuromodulatory information by differentiatingbetween serotonin (5-HT) and norepinephrine sensitive behavior categories. Head movement is a newlycharacterized behavior endpoint in the FST and TST with a selective 5-HT sensitivity. In this investigation,we show that the baseline and drug response profile of head movement previously found in the 129S6strain of mice (Lockridge et al., 2010) is reproducible in the C57 strain. Head movement is inverselycorrelated to FST swimming and elevated in the TST by SSRI administration. The use of a weighted bin

ntidepressantSRIerotonin subtype receptorin sample analysis

sample analysis method differentiates TST behaviors into fluoxetine-responsive head movement anddesipramine-responsive struggling. The use of 5-HT subtype receptor agonists, after depleting endoge-nous 5-HT with pCPA, shows the head movement suppressing effect of 5-HT2A and 5-HT2C postsynapticreceptor activation. 5-HT1A and 5-HT1B agonists were ineffective. We propose that a head movementfocused analysis can add sensitive and reliable 5-HT detection capability to mouse TST testing withminimal effort but significant reward.

. Introduction

As a tool for preclinical research on mood disorders, the forcedwim test (FST) and the tail suspension test (TST) are popular,fficient and minimally traumatic screens for antidepressant andepression related activity. Both tests place the subject, often a

odent, in a situation of inescapable stress that aims to be moresychologically than physically threatening. The frequency anduration of struggling exhibited during the period of entrapment

∗ Corresponding author at: Department of Neuroscience, University of Minnesota,-145 Jackson Hall, 321 Church St. SE, Minneapolis, MN 55455, USA.el.: +1 612 625 8613; fax: +1 612 626 5009.

E-mail addresses: yuanx033@umn.edu, yuanx033@gmail.com (L.-L. Yuan).

166-4328/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.bbr.2013.02.032

© 2013 Elsevier B.V. All rights reserved.

is broadly responsive and selective to a variety of antidepres-sant treatments [1–3] although the psychological interpretationof immobility and struggle patterns under stress is an unresolveddebate [2,4,5].

The FST, in which the subject is confined to a small pool ofdeep water, was the first of these tests to be developed [1] andhas undergone multiple procedural and analytical modificationsincluding a differentiation of active behaviors into serotonergicallymodulated swimming and noradrenergically modulated climbingcategories [6,7]. This type of mechanistic specificity is highly valu-able but the test itself comes with some disadvantages including

the risk for hypothermia, ear tag interference, water aspiration andstress-induced hind limb paralysis in some mouse strains [8,9]. Bycontrast, the TST is simpler, more drug sensitive and more reliable,particularly in response to selective serotonin reuptake inhibitors

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SSRIs) [8]. However, the TST, which relies on a generic strugglingategory, has traditionally been unable to differentiate betweenlasses of antidepressants.

In a previous paper, our lab examined head movement as aotential new TST/FST behavior analysis category that was selec-ively responsive to the SSRI fluoxetine in129SvEv male mice [9].n the current study, we sought to confirm these results in the

ore common C57Bl/6J mouse strain. Furthermore, we investi-ated the potential of head movement to provide TST analysis withpecific mechanistic details on 5-HT modulation of antidepressantction.

. Materials and methods

.1. Animals

C57Bl/6J male mice imported or bred from mice obtained from Jackson labsere behavior tested at 7–12 weeks. All mice were housed in conventional facilities

n a 12:12 light cycle with ad libitum access to food and water in groups of 3–4xcept during experiments when each animal was singly housed. Animals wereemoved from analysis for improper injections, visible injury or tail-climbing duringuspension. In the 5-HT subtype receptor agonist experiment, mice with a veryow naïve baseline for head movement (≤20 s cumulative duration) were removedrom analysis based on preliminary results indicating dramatic behavior variabilitymongst this group during subsequent testing. Except where explicitly stated in theext, a unique cohort of animals was used for each treatment group in the FST andn the TST and each animal received only a single test. The use of animals for thesetudies was approved by the University of Minnesota Institutional Animal Care andse Committee.

.2. Behavior tests

The FST protocol was adapted from previous descriptions [10,11]. Mice werelaced in clear plexiglass cylinders (20 cm tall × 15 cm wide) filled with tap watero a height of 14 cm and maintained at a temperature of 25 ± 1 ◦C for a single 5 mineriod.

In the TST mice were suspended for 5 min on a hook by tape wrapped aroundhe distal third of the tail such that the body hung 8 cm from the support pole andhe nose 5 cm from the base of the apparatus [12]. During initial TST testing, some

ice were removed from analysis due to tail climbing. To avoid this, we outfittedubsequent mice with a 1–2 inch flexible plastic cone cut from the bottom tip of aecapiCone Disposable Mouse Restrainer (Braintree Scientific Inc., Braintreee, MA,SA). The cone was placed around the base of the tail with the larger end opening

ust below the tape. Treatment conditions were compared to controls that wereested using the same procedural details.

.3. Drugs

All drugs were obtained from Sigma (Sigma–Aldrich, St. Louis, MO, USA) andissolved into 0.9% saline proportioned for an average injection volume of 0.3 mL for

25 g mouse with the exception of sertraline and citalopram, which were calculatedor a 0.6 mL average to ensure complete solubility. Fluoxetine despiramine were

ade fresh on the day of testing. Sertraline, citalopram, buspirone, CGS-12066A,OI, and RO 60-0175 were made as stock at the in-use concentration and stored

n aliquots at −20 ◦C and thawed the day of use. 4-chloro-dl-phenylalanine methylster (pCPA) was made fresh before each daily injection.

Antidepressants or saline were administered by intraperitoneal (ip) injection0 min prior to FST or TST testing in the following concentrations: desipramine20 mg/kg), fluoxetine (30 mg/kg), sertraline (5, 10, 20 mg/kg) or citalopram20 mg/kg). Each animal received one drug and a single session of one test type.rior to testing with serotonin (5-HT) subtype receptor agonist drugs, mice wereretreated with pCPA to deplete systemic 5-HT and reduce endogenous compe-ition for post-synaptic receptor activation. Mice first received a naïve 5 min TSTollowed by 4 consecutive days of ip injections with pCPA (100 mg/kg) and a secondST, 15 min after the 4th injection. On the 5th day, animals received one of 4 5-HTubtype receptor selective agonists: Buspirone (5-HT1A, 3 mg/kg), CGS-12066A (5-T1B, 2 mg/kg), DOI (5-HT2A, 1 mg/kg) or RO 60-0175 (5-HT2C, 4 mg/kg) with a finalST 30 min after this injection.

.4. Video preparation

All behavior tests were video recorded during testing and analyzed later.

ideos were imported from analog tape then exported onto a computer hardrive using iMovie HD (Apple Inc.). Each file was then split into clips usinghe SplitFuse program (Likely Software). Each clip was viewed by one of multi-le blind observers and assigned a behavior category based on detailed writtenefinitions.

Research 246 (2013) 168– 178 169

2.5. Behavior category definitions

2.5.1. FSTClimbing (C): Primarily vertical movement of the forelimb/s against the wall.

This can also include circular, diagonal or horizontal movements against the wall aslong as the force is clearly directed at the wall itself.

Swimming (W): Movement of the hind limbs or tail resulting in a propulsiveforce. Often but not always accompanied by translocation. Includes movementaround the perimeter of the pool, across the pool, and towards the wall if no fore-limb movement is present and the presence of the wall is the only obstacle totranslocation.

Immobility (I): No active movement occurring (except for twitches, shivers, orcorrective wall-bouncing). The mouse’s body may be parallel or perpendicular tothe surface of the water or somewhere in between. The head may be above thewater or level with it. The body may be in the center of free water or pressed againstthe wall. The mouse may be completely still or moving through the water withoutactive maintenance of movement.

Head movement (H): Extension of the head either across the surface of the wateror elevated above the surface, head swivel up-and-down or side-to-side, sniffing.These behaviors can occur together or separately. Identify sniffing by nose and/orwhisker movement.

2.5.2. TSTStruggling (S): Large amplitude, lasting, whole limb or body movement. Twist-

ing, shaking, raising of the body and bicycling in the air with the limbs. Any bodymovement that moves from a pivot point near the tail or during which the tail isalso moving is included. The presence of hind limb movement automatically scoresas struggling. Rapid, non-sporadic fore limb activity also falls into this category.

Immobility (I): No active movement occurring (except for sporadic, short-duration twitches or shivers). The body may point straight down or be curled upwarddepending on the animal. The mouse may be swinging from previous motion butno active maintenance of movement should be taking place.

Head movement (H): Elongation of the neck to extend the head, head swivelup-and-down or side-to-side, sniffing and grooming. These behaviors can occurtogether or separately. Identify sniffing by nose and/or whisker movement. Half-body movements do not exclude head movement, especially if they appear to be inservice of head movement positioning or adjusting sniffing range. Grooming shouldinclude regular movement of the paws up over the nose.

2.6. Behavior analysis methods

2.6.1. Standard bin sample codingThis analysis was based on a previously published method [6]. Each video was

split into 60 5 s clips. Clips were opened in one-minute groups but viewed in a ran-dom order within that group. The observer initially viewed the first 1 s of the clip andassigned a score if a single behavior clearly dominated the entire second. If no dom-inant behavior emerged, the next second was evaluated. If behaviors were similarlymixed, the entire clip was viewed and the dominant behavior was scored regardlessof the actual duration of any one episode of behavior. Behaviors sometimes occurredin equal mix, in rapid alternation or even simultaneously so a hierarchy was usedto resolve clip identification in these cases: C > W > H > I (FST) or S > H > I (TST). Thisdecision was based on our belief that immobility should represent a true absence ofdirected/intentional activity but that head movement, as a relatively unknown cate-gory, should only be scored when a more traditional behavior could not be identifiedfirst.

2.6.2. High resolution codingEach video was split into 300 1 s clips, opened 20 at a time and viewed in a

random order within that group. The whole clip was viewed and scored if a dom-inant behavior could be observed to occur for more than 0.5 s. If two behaviorswere present in equal duration, each half-second was given a different score. Thesame hierarchical category preferences were used to resolve issues of simultaneousbehaviors. We assumed the same order characterized energy expenditure, withclimbing/struggling as maximally energetic and immobility minimally so, based onour observations of the physical vigor engaged during each activity type (Supple-mental Fig. 1).

2.6.3. Weighted bin sample analysisThis analysis was only used for TST videos after basic head movement behaviors

had been established and characterized using standard and high resolution analysismethods. The bin sample technique was the same as described in the standard sys-tem but the category hierarchy was rearranged to place head movement ahead ofstruggling. In clips where head movement and struggling occurred simultaneouslyor in equal measure, head movement was scored preferentially (Fig. 1).

2.7. Statistics

Formal statistical analysis was conducted in MATLAB using a multiway (n-way)analysis of variance (ANOVA) that included weight as a variance factor as well asexperimentally-dependent factors such as drug treatment. When significant factors

170 A. Lockridge et al. / Behavioural Brain

Fig. 1. Video demonstration of TST behaviors. Clips from videos taken during thesrw

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ertraline dose-response experiment demonstrate head movement behavior occur-ing in isolation and simultaneously with struggling behavior. A clip of strugglingithout head movement is shown for comparison.

ere identified, we employed the multiple comparison test function on the ANOVAutput to identify dissimilar groups under each factor condition. In some cases Stu-ent’s t-test (two-tailed, paired or unequal variance as appropriate to context) washen used to obtain p-values for specific pairs. Significance threshold was set at

< 0.05. All reported uncertainties and graph error bars refer to standard error ofhe mean.

. Basic characterization

.1. Interstrain comparison

In a previous paper, our lab characterized head movement in29SvEvTac (S6) male mice [9]. The first step in our current inves-igation was to use a standard bin sampling analysis (Section 2.6.1)o indentify similar behaviors in C57Bl/6J male mice in the FST

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Research 246 (2013) 168– 178

and TST for comparison. C57Bl/6 J (10) and 129S6 mice (19) dis-played a nearly identical distribution of behavioral categories inthe FST 30 min after a saline injection (Fig. 2A). Immobility com-prised 20–30% of time, climbing 10–15% and swimming 50–65%.Head movement occurred in the absence of other activities 6% ofthe time at 18 ± 5 s 129 vs. 17 ± 6 s C57 (F(1,28) = 1.48, p = 0.24). Weinitially experienced difficulty with TST testing in the C57 strain asseveral mice climbed their tails, a behavior that was not seen in 129mice. This observation has been made previously, by other inves-tigators, particularly for the C57Bl/6 genetic background [8,13]. Inthis set of tests, mice that climbed their tails were eliminated fromsubsequent analysis. Among the remaining individuals, C57 mice(8) showed more TST immobility, 114 ± 11 s (38% time) C57 vs.76 ± 6 s (25% time) 129 (F(1,23) = 10.27, p < 0.01) and less head move-ment 6 ± 3 s (2%) C57 vs. 33 ± 5 s (11%) 129 (F(1,23) = 14.16, p < 0.01)than previously found in 129 mice (16) following saline injection(Fig. 2B). Struggling was similar for both strains at ∼60% of totaltime. However, the relative dominance of behaviors within the testwas similar between strains (See [9] for original report of 129 data).

We also examined the minute-by-minute temporal profileof C57 head movement in both tests. As expected, immobilityincreased over time while active behaviors decreased in both theFST (Fig. 2C) and the TST (Fig. 2D), with the exception of swimming,which increased in the second minute and then decreased slowly(2C). By contrast, head movement remained at a steady low baselinethroughout the 5 min duration of either test with a linear trendline

slope of 0.1 in the FST (2C) and 0 in the TST (2D). The failure to mimicthe temporal pattern of any other behavior supports the hypothe-sis that head movement is an independent category rather than asubcomponent of one of the traditional behavior classifications.

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bited a similar distribution of behaviors in the forced swim test (FST) following a and a smaller cumulative duration of head movement. (C) Head movement in theined by minute. (D) TST head movement had a similar small amplitude but steady

viors. All behavior was analyzed using the 5 s standard bin sampling method. Data panel A and B legends. **p < 0.01 vs. adjacently presented value.

Brain Research 246 (2013) 168– 178 171

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Fig. 3. High-resolution analysis of behavior state transitions in C57 mice. (A)Head movement occurred more often during FST two-state transitions of risingenergy, particularly preceding swimming. (B) During three-state FST transitions,head movement showed a preference for bridging low energy immobility to higherenergy swimming but little association to climbing. (C) A similar analysis of TSTtwo- and three-state behavior transitions also shows a pattern of elevated headmovement frequency during transitions of increasing energy. All behavior was ana-

A. Lockridge et al. / Behavioural

.2. State transition energy preference

It could still be argued that head movement is a subcompo-ent of one of the active behaviors if it is a weak form thatccurs primarily during physical exhaustion. We therefore used aigh-resolution, continuous half-second analysis method on videosf the same C57 mice to more precisely define the individualccurrences of head movement as it related to the other behav-ors (Supplemental Fig. 1). We focused on transitions from oneehavior state to another and whether this shift represented aove from higher to lower energy or the opposite, based on

ur assumption of hierarchical energy expenditure (see Section.6.2). In the FST, two-state transitions in which head movementreceded swimming occurred more frequently than those withwimming occurring first, 8.4 ± 1.9 HW vs. 4.4 ± 1.7 WH transi-ions (F(1,19) = 6.21, p < 0.05) (Fig. 3A). 3-state transitions with head

ovement bridging between two dissimilar behavior states alsoound a preference for rising energy transitions between immo-ility and swimming, 4.3 ± 1.2 IHW vs. 1.0 ± 0.3 WHI transitionsF(1,19) = 15.34, p < 0.01) (Fig. 3B). Head movement and climbingccurred together infrequently and with even distribution (3AB).n our previous work with 129 mice, head movement also showed

stronger association with swimming than climbing and signif-cantly preferred increasingly energetic transitions [9]. A similarattern of preference was observed in TST testing for both C57nd 129 mice. C57 mice more often displayed head movementollowing immobility than preceding it, 6.3 ± 1.2 IH vs. 3 ± 0.8I transitions (F(1,15) = 8.81, p < 0.05) and as a bridge between

ow effort immobility and high effort struggling, 4.9 ± 1.0 HIS vs..0 ± 0.6 SHI transitions (F(1,15) = 18.29, p < 0.01) (Fig. 3C). In gen-ral, head movement more frequently represented a shift towardslevated energy expenditure, the opposite to our expectations ifhe behavior were predicated on a state of physical exhaustion. Theharacteristics of saline-injected mice of two strains support headovement as a novel independent behavior category.

. Serotonergic sensitivity

.1. Head movement in FST

FST swimming has been well validated as a behavior withominant serotonergic (5-HT) modulation in mice [6]. The prefer-ntial association between head movement and swimming in theransition state analysis prompted us to look more closely at theeurochemical controls of this behavior. Based on similar obser-ations in 129 mice [9], we included 5-HT and norepinephrineelective antidepressants in the FST and TST as parallel treatmentroups to the mice already described. Drawing from the high-esolution analysis, we plotted FST individuals by their swimmingnd head movement scores to reveal a moderate inverse correlationmong saline-injected controls (R2 = 0.43, linear trendline) (Fig. 4A).his correlation intensified (R2 = 0.88, linear trendline) among ani-als treated with the selective serotonin reuptake inhibitor (SSRI),

uoxetine (30 mg/kg ip, n = 9) (4A). No correlation was observedetween head movement and climbing either in the saline groupR2 = 0.17, linear trendline) or in animals treated with the selec-ive tricyclic desipramine (20 mg/kg ip, n = 9) (R2 = 0.01) (Fig. 4B).he lack of interaction with climbing, a norepinephrine modulatedehavior, and desipramine, which is dominantly active on nora-renergic transmission, serves as a strong control highlighting thepecificity and consistency of head movement response to 5-HT

ctivity. The proportional scatter among individuals is interest-ng because although both swimming and head movement wereeactive to fluoxetine, some individuals primarily increased headovement (n = 7, 241% increase vs. saline average) while others

lyzed using the half-second high-resolution analysis method. FST (N = 10), TST (N = 8)occurred 30 min after a saline injection. *p < 0.05, p < 0.01 vs. adjacently presentedvalue.

increased swimming (n = 3, 133% increase vs. saline average). Itcould be that these behaviors reflect the dominance of different 5-HT subsystems or receptor types. With additional characterization,we hypothesize that the differentiation between head movementand swimming might be useful to explain some of the scatter in FSTbaseline and control behavior profiles. Furthermore, the divergentresponses might be useful in understanding the neuromodula-tory or genetic background factors that influence antidepressanttreatment efficacy differences between depressed individuals, par-ticularly among various SSRIs [14].

4.2. Head movement in TST

Although there is a potential for a head movement categoryto refine FST analysis, for the purpose of this investigation we

172 A. Lockridge et al. / Behavioural Brain Research 246 (2013) 168– 178

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Fig. 4. Selective association between head movement and serotonin (5-HT) sys-tem. (A) A moderate inverse correlation was seen among saline-injected animalsbetween head movement and 5-HT modulated swimming in the FST (solid line). Thiscorrelation was greatly strengthened and shifted towards higher head movementin animals treated with the SSRI fluoxetine (30 mg/kg; dashed line). (B) No correla-tion was found between head movement and norepinephrine-modulated climbingfollowing either saline (solid line) or the norepinephrine-selective desipramine(r

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Fig. 5. Comparison of TST behaviors by three analysis methods. (A) All 3 analysismethods presented a similar drug response pattern but weighted 5 s bin samplingelevated head movement baseline by prioritizing this category over struggling dur-ing simultaneous behavior. (B) Bin weighted analysis did not interfere with theability of both fluoxetine and desipramine to significantly reduce immobility butstruggling was now selectively responsive to desipramine. (C) Weighted codingincreased head movement uniformly throughout the test, leaving the temporal pro-

20 mg/kg; dashed line). All behavior was analyzed using the half-second high-esolution analysis method. N numbers are shown in the legends.

hose to focus on head movement in the TST where no neuro-odulatory specificity is currently available. Using standard bin

ampling, we confirmed that fluoxetine (9) increased head move-ent cumulative duration to 21 ± 4.1s from 6 ± 2.5 s saline (8)

F(2,25) = 7.96, p < 0.01) while desipramine had no effect (Fig. 5A).e also introduced a new weighted analysis method that we hopedould elevate baseline head movement (Section 2.6.3). In both

tandard and high resolution coding, we resolved simultaneousehaviors by valuing all other active behaviors above head move-ent in order to characterize this novel category in a context most

imilar to traditional analysis methods. However, this left us with very low baseline, particularly in the C57 strain, that exaggeratedhe impact of error and made any study of bidirectional modula-ion very difficult. So, we reversed the hierarchy for simultaneousctive behavior resolution to H > S > I, separating instances of strug-ling with head movement from those without (See Fig. 1). Theeighted analysis did universally elevate the baseline to 64 ± 8.2 s

aline, 102 ± 12.4 s fluoxetine and 59 ± 8.6 s desipramine and main-ained the relative drug treatment effects (F(2,25) = 3.69, p < 0.05)Fig. 5B). Immobility was still significantly reduced by both antide-ressants (F(2,25) = 7.72, p < 0.01) and, interestingly, struggling wasow enhanced only by desipramine (F(2,25) = 6.8, p < 0.01) suggest-

ng that struggling might represent a noradrenergically selectiveehavior in this analysis (Fig. 5B). Relative behavior distributionver time remained largely unchanged from the pattern seen in

ig. 2D. The immobility trendline had a rising slope (5.2) and strug-ling, a diminishing one (−5.1) (Fig. 5C). Head movement wasargely stable over time (−0.1 slope of linear trendline) but the-intercept was shifted from 1.25 to 13.25 (5C).

file of TST behaviors relatively unchanged. N numbers are shown in the panel Blegend. *p < 0.05, **p<.01 vs. saline unless otherwise indicated.

4.3. SSRI response

Looking to generalize our findings beyond fluoxetine, we testeda new set of C57 mice with the SSRIs sertraline (20 mg/kg ip,n = 10) and citalopram (20 mg/kg ip, n = 9) and new saline con-trols (7). There was a significant effect of treatment with sertralineand citalopram increasing head movement from 54 ± 9.9 s saline

to 97 ± 15.6 s and 96 ± 15.4 s, respectively (F(2,25) = 5.55, p < 0.05)(Fig. 6A). The ANOVA multicompare function identified salineand sertraline as significantly different but post hoc t-tests gave

A. Lockridge et al. / Behavioural Brain Research 246 (2013) 168– 178 173

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Fig. 7. Dose-response curve of sertraline. (A) Head movement was tightly respon-sive to changes in sertraline dose, significantly increasing total behavior durationabove control level at 10 and 20 mg/kg. (B) Immobility trended downwards withincreasing sertraline dose but large group error contributed to a lack of significantchange. (C) Struggling was unresponsive sertraline dose. All behavior was analyzed

5.1. pCPA

s expected but had no effect on struggling (C). N numbers are shown in the figureegend. *p < 0.05 indicates significance versus saline treatment group.

values below 0.05 for both sertraline (0.034) and citalopram0.037). ANOVA also identified both SSRIs as significantly reducingmmobility (F(2,25) = 4.63, p < 0.05) but having no impact on strug-ling (p = 0.1436) (Fig. 6BC). This was the first experiment in whiche began placing soft, flexible plastic cones around the base of eachouse’s tail to prevent them from climbing it. This technique was

ighly effective and used in all subsequent TST tests.To further probe the flexibility and sensitivity of the head move-

ent response, we generated a sertraline dose response curve. Wedministered the following doses and group numbers: saline (3),

mg/kg (9), 10 mg/kg (8), 20 mg/kg (2). There was no significantffect of experimental iteration on saline behaviors so these dataere combined with previous saline controls for a group total of

0 mice. A similar analysis of 20 mg/kg treated mice showed thatead movement results from this group was not dissimilar fromny other experimental run and the data were combined for a finalroup number of 12. Sertraline dose was a significant factor in

ead movement responses F(3,38) = 5.15, increasing total duration

rom 51.50 ± 7.71 s (saline) to 74.44 ± 7.19 s (5 mg/kg), 95.0 ± 6.12 s10 mg/kg) (p < 0.001 vs. saline) and 100.42 ± 13.26 s (20 mg/kg)

using weighted 5 s bin sampling analysis. N numbers are shown parentheticallyalong the horizontal axis. **p < 0.01, ***p < 0.001 indicates significance versus salinetreatment group.

(p < 0.01 vs. saline) (Fig. 7A). Immobility responded insignificantlyto dose (F(3,38) = 1.66, p = 0.19) with a downward trend but largeerror bars for each group average (Fig. 7B). Struggling was largelydose-unresponsive (F(3,38) = 0.45, p = 0.72) (Fig. 7C).

5. Post-synaptic 5-HT receptors

Reaching beyond general 5-HT sensitivity, we wanted to probewhether TST head movement was more responsive to some sub-type receptors than others. We focused on 5-HT1A, 5-HT1B, 5-HT2Aand 5-HT2C, based on popular mention in depression related liter-ature [15,16].

Seeking to ensure a high specificity of drug action in this exper-iment, we first treated mice with the tryptophan hydroxylase

174 A. Lockridge et al. / Behavioural Brain Research 246 (2013) 168– 178

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Fig. 8. Involvement of 5-HT receptor subtypes in head movement. 5 days after a naïve TST, and 24 h after a final pCPA injection (100 mg/kg for 4 days) to deplete endogenous5-HT + 2nd TST, mice received a selective receptor agonist and a 3rd TST. (A) Buspirone (5-HT1A, 3 mg/kg) nudged head movement downward but not significantly. (B)CGS-12066A (5-HT1B, 2 mg/kg) had no effect on head movement. (C) DOI (5-HT2A, 1 mg/kg) suppressed head movement significantly as did Ro 60-0175 (5-HT2C, 4 mg/kg)( bers as

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D). All behavior was analyzed using weighted 5-sec bin sampling analysis. N numignificance vs. naïve value for the same group.

nhibitor pCPA (100 mg/kg ip × 4 days) to deplete systemic 5-HT.ur intention was two-fold: to determine whether low stores ofvailable 5-HT would impact TST head movement and to elimi-ate endogenous competition for receptor binding sites. Naïve mice33) were administered a TST test before treatment and 15 minfter the last pCPA injection (Section 2.3). We did see a signifi-ant shift in behaviors in the second test as compared to the firstor the entire set of treated animals although the magnitude ofhange was mild, ∼20%, compared to the anticipated 60% loss ofrain 5-HT that has been reported following a similar procedure17]. Head movement (F(1,63) = 5.63, p < 0.05) (Fig. 9A) and strug-ling (F(1,63) = 5.77, p < 0.05) (Fig. 9C) were both diminished, pushingmmobility higher (F(1,63) = 11.14, p < 0.01) (Fig. 9B). It was some-

hat unexpected to observe an effect of pCPA alone as the lack ofreatment effect has been noted in numerous other mouse stud-es examining immobility in the TST [18–20] or FST [15,21–23]. It isossible that our modified analysis was more sensitive but it seemsore likely that this effect arises from comparing the same mice in

heir naïve and secondary TST exposures while the reported stud-es used saline-injected side-by-side controls. Group size may alsoe a factor as pCPA did not represent a significant deviation fromaseline when head movement was analyzed within subgroups of–9 mice, divided into separate 5-HT receptor agonist treatmentroups (Fig. 8).

.2. 5-HT1A

To investigate the potential modulatory role of 5-HT1A on headovement, we used the agonist buspirone (3 mg/kg, n = 7), at a

ose that has been shown to increase FST immobility in mice [24].uspirone appeared to encourage a decrease in head movement

rom 62 ± 5.3 s naïve to 40 ± 7.4 s but this was not significant inhe ANOVA analysis (F(2,20) = 1.97, p = 0.1759) (Fig. 8A). Given the

re shown parenthetically in the header for each panel. *p < 0.05, ** < 0.01 indicates

direction of the trend, we cannot rule out that a larger group sizemight reach the threshold of significance.

5.3. 5-HT1B

The 5-HT1B agonist CGS-12066A (2 mg/kg) has been shown todecrease FST immobility in mice [16]. We used a similar dose24 hours after the last pCPA treatment (n = 9) but no change in headmovement was observed (F(2,26) = 1.34, p = 0.2854) (Fig. 8B).

5.4. 5-HT2A

We were particularly interested in the results of our 5-HT2Atesting, as this receptor has been speculated to be involved in thefew published references to FST behaviors that sound somethinglike head movement (see Section 6.1) [25,26]. We relied on thewell-established agonist DOI (1 mg/kg, n = 8) to show that 5-HT2Aagonism significantly reduced TST head movement from 63 ± 13.8 snaïve to 33 ± 10.0 s (F(2,23) = 4.13, p < 0.05) (Fig. 8C). If elevated headmovement is indicative of antidepressant effect, as suggested bythe SSRI results, then a suppression of head movement through 5-HT2A agonism is consistent with other studies that have implicateddiminished 5-HT2A receptor activity in both short and long-termantidepressant effects [22,27,28].

5.5. 5-HT2C

Our 5-HT2C agonist, Ro 60-0175 (4 mg/kg, n = 8), was the mosteffective in suppressing head movement from 66 ± 5.9 s naïve to

29 ± 8.9 s (F(2,23) = 6.93, p < 0.01) (Fig. 8D). Ro 60-0175 has notshown a consistent impact in the available published data on FSTbehavior, with some reports indicating antidepressant effect [29],no effect or efficacy only in boosting SSRI impact [30]. Therefore, we

A. Lockridge et al. / Behavioural Brain

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Fig. 9. Effects of 5-HT subtype receptor agonists on all TST behaviors. Data is pre-sented as the ratio of treatment condition over the naïve, pre-treatment, value in anindividual then averaged by treatment group. (A) Fig. 6 data is summarized showingdecreased head movement following whole group pCPA and subgroup DOI or Ro 60-0175. (B) pCPA and Ro 60-0175 produced higher immobility ratios and diminishedstruggling changes (C) but other treatments were ineffective. The line at Y = 1.0 ineach panel represents the level at which no treatment effect would be expected. Allbehavior was analyzed using weighted 5 s bin sampling. N numbers are presented intvt

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he figure legend at the top. *p < 0.05, **p < 0.01 indicates significance for treatments. naïve, pre-treatment as calculated based on the duration values used to producehe ratios.

hose a dose that was at least below the threshold for locomotionmpairment [30] that might have confounded data interpretation.n our case, Ro 60-0175 produced a clear depressant effect (see alsoection 5.7 and Fig. 9).

We also note that pCPA appeared to have a moderately dimin-shing effect in this subgroup with head movement at 43 ± 6.3 sn the pCPA treated group. However, ANOVA did not identify thisroup as significantly different from pre-treatment TST behavior.

.6. Other TST behaviors

Summarizing multiple panels in Fig. 6, pCPA, DOI and Ro0-0175 all reduced head movement as a ratio of naïve base-

ine, calculated for a single mouse then averaged over similar

Research 246 (2013) 168– 178 175

treatment conditions (Fig. 9A). Buspirone produced a downwardtrend that was not significant for the group size tested. Further-more, the individual scatter was generally smallest for the headmovement behavior category. Differences in scatter were particu-larly notable in the DOI treatment group where statistical analysisidentified weight as a significant interacting and independent fac-tor (F(6,23) = 6.12, p = 0.0021). Upon closer inspection, a single mouseat 26 g appeared to be the source, displaying behaviors 200%removed from the group averages in several cases. Error bars arelargely diminished with this mouse removed from analysis but thedirection of the average and statistical significances are unchangedand the data in Figs. 8 and 9 are presented with this mouse includedin all relevant data sets. Ro 60-0175 increased immobility to aratio of 1.49 ± 0.05 of naïve (F(2,23) = 8, p < 0.01 based on duration)and pCPA increased it to 1.22 ± 0.06 (see 5.2) (Fig. 9B). Strugglingchanges ran contraphase to immobility with Ro 60-0175 values ata ratio of 0.43 ± 0.10 under naïve (F(2,23) = 5.94, p < 0.05 based onduration) and pCPA at 0.86 ± 0.16 in the same direction (see Sec-tion 5.2) (Fig. 9C). The standout in this analysis was the effect of5-HT2A agonist DOI on head movement, which was not observedin either of the traditional struggling or immobility behavior cate-gories. Combined with noticeably smaller error in most treatmentgroups, this is consistent with our dose-response data suggestingthat head movement may be a more sensitive and more reliablebehavioral endpoint for evaluating serotonergic activity, particu-larly 5-HT2 receptor activation.

6. Discussion

6.1. Rodent head movement in the literature

We mentioned that some authors have referenced FST behaviorsthat might be similar to our head movement category (see Section5.5). Following chronic restraint stress, rats have been observedto increase sniffing and head elevation compared to desipramine-treated animals [31]. “Swimming induced head twitching” wasfound to be enhanced in rodents pre-exposed to overcrowdingstress although the twitches were unaffected by treatment with5-HT specific drugs [25]. A review on the non-selective antidepres-sant imipramine found that this drug reduced head-shakes in theFST and also suggested 5-HT2 receptors as a likely mediator, point-ing to increased shakes in females during periods of high circulatingestrogen, which is known to increase 5-HT2 binding site receptordensity [26]. A more recent study categorized head shakes as a non-selective antidepressant component reduced by imipramine andsharing a predictive correlation with swimming activity [32]. Giventhe short descriptions of these behavior phenomena, it is difficult todetermine if any of these refer to the same activity we have char-acterized. Outside of the FST context, studies on risk assessmentbehaviors have linked investigatory sniffing with 5-HT transmis-sion [33]. We have not found any prior description of TST headmovements.

The use of the drug DOI in this study to manipulate head move-ment calls to mind the nominally similar head twitch response,which is a behavior test commonly induced by DOI administra-tion. 5-HT2A modulation of this behavior, however, is dissimilarfrom the patterns we have observed. Head twitches are inducedby activating 5-HT2A receptors and can be partially blocked byantagonizing the receptor [34]. By contrast, head movement herewas diminished by DOI treatment and we previously showed adramatic increase in head movement by antagonizing the 5-HT2A

receptor with ketanserin prior to antidepressant treatment in atransgenic mouse [9]. Futhermore, head twitches appear sporadic,small amplitude and uncontrolled whereas the head movement wehave characterized involves larger amplitude full head swivels and

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ertical movements from the neck that can last continuously forver a second. Combined with nose/whisker activity that is identi-al to the appearance of sniffing, head movement behaviors givehe impression of deliberate environmental evaluation throughensory rather than active exploration. While this motivationalttribution is entirely speculative, the impression is certainly notne of involuntary muscle spasms. Nevertheless, DOI head twitchesponse has been suggested as an independent 5-HT2A behav-oral screen for use in mood disorder research previously [35]. Thedvantage of using the methods we describe to assess serotoner-ic activity include the ability to test mice with or without theddition of exogenous drugs, an impossibility for head twitches,nd to obtain these results in parallel with immobility, which as alassic depression-related behavior category has a rich history ofackground literature to support interpretation.

If head movement has been present as an unrecognized com-onent of behavior in the TST and FST, how was it being analyzedreviously? For the TST, this likely depends on whether you areeasuring jerk force on a pressure-sensitive spring and what

hreshold you set for struggling detection. Certainly instancesf simultaneous struggling and head movement would only beefined as struggling. Head movements occurring while the restf the body is still would likely be scored as immobility unless theensitivity threshold was set very low. FST descriptions of immobil-ty often include a phrase like “motionless except for movementsecessary to keep the head above water”. This description suggestshat the head movements we have isolated during FST analysisere likely to have been scored as immobility in labs using this

tyle of definition. However, this description of immobility does notatch our experience as we have never seen a mouse’s head drop

elow the line of the water during periods of immobility despiteemaining truly motionless, occasionally for long durations. In fact,y grouping head movement with immobility, two behaviors thate have shown to often act in opposition, the analysis may lose

ome of the sensitivity offered by a more strict definition of immo-ility. This is an apt time to note that while we have used a 5 min testnd analysis, many labs prefer to evaluate the last 4 min of a 6 minest [2]. This decision was based on our interest in active behav-ors, which dominate the first 2 min of these tests. Regardless, theemporal stability of head movement suggests that baseline andelative drug response for this category is likely to be the same forither procedural method.

.2. Serotonergic modulation of head movement

Acute stress triggers the release of 5-HT into multiple brainreas, notably the hippocampus and prefrontal cortex, where itay activate any of 14 different classes of serotonin receptor sub-

ypes [15,33,36]. Activation of each receptor subtype initiates aistinct intracellular signaling cascade that ultimately influencesoth serotonergic transmission properties and secondary targetsuch as other neurotransmitter systems or neuroplasticity mecha-isms [37]. Of these, 5-HT1 and 5-HT2 type receptors have receivedhe most attention in the realm of depression research including

odulation of FST/TST behaviors and SSRI antidepressant response15,38–41]. Mild to moderate 5-HT release is thought to firstctivate high affinity 5-HT1 receptors while greater and more pro-onged concentrations are required to engage the low affinity 5-HT2eceptors [42,43]. 5-HT1 receptors may be presynaptic or postsy-aptic, but their function as presynaptic inhibitory autoreceptors

s believed to dominate stress and antidepressant response [15].y contrast, postsynaptic 5-HT2 receptor activation depolarizes the

ells and predominantly enhances excitatory activity [44,45]. Tohis extent, their actions on 5-HT transmission are oppositional andikely to regulate distinct and dissimilar aspects of depression andrug response [44].

Research 246 (2013) 168– 178

5-HT1A receptors are heterogeneous, operating as both postsy-naptic and presynaptic inhibitors of neural activity in the receptiveand firing cell, respectively. Our current findings suggest that post-synaptic 5-HT1A activation does not contribute significantly to headmovement modulation. Buspirone acts at both pre- and postsynap-tic locations but because we depleted 5-HT availability with pCPA, itis unlikely that autoreceptor activation could meaningfully impact5-HT release. We assume, therefore, that any drug effects, or lackthereof, were primarily a result of postsynaptic activation, whichhas been implicated in the antidepressant action of some 5-HT1Aagonists [24]. The absence of a strong head movement response to5-HT depleted busiprone treatment does not rule out the poten-tial impact of presynaptic receptor activity. A great deal of researchhas characterized the significance of negative feedback from thepresynaptic 5-HT1A autoreceptor in mediating anxiety, depressivedisorders and SSRI efficacy (e.g. [46] for review). We did not findany evidence that 5-HT1B receptors were involved in controllinghead movement behavior.

Both 5-HT2A and 5-HT2C agonists were effective in suppress-ing head movement. In combination with the robust SSRI-inducedincrease in head movement, these data are in agreement with otherstudies that have linked some SSRI efficacy to 5-HT2 receptor antag-onism [44,47]. In our previous publication, head movement wasdramatically elevated when ketanserin was used to block 5-HT2receptors before fluoxetine treatment [9]. From this, we expecthead movement to be higher in less stressful circumstances where5-HT neurotransmission occurs at smaller concentrations, belowthe threshold of 5-HT2 activation and diminished as stress and 5-HTtransmission increases. We observed the converse as 5-HT2C acti-vation by Ro 60-0175 increased immobility, a sign of higher stressthat was correlated to decreases in both struggling and head move-ment. This argument appeals to the somewhat anthropomorphizedimpression of this behavior as “looking around” and investiga-tory. Environmental evaluation could provide valuable informationtowards deciding on a resource-conservation vs. force-activationstrategy of escape if the perceived threat to survival is indirect orminimal enough to allow time for a sensory analysis.

6.3. Advantages of a modified TST analysis

We have shown that head movement is an isolatable componentof the FST and have focused on developing it for use in the TST. Thisis partly because the modified FST includes climbing and swimmingbehavior categories that already allow it to differentiate betweendrugs and conditions with distinct modulatory impacts [6] whilethe TST has heretofore been a generic screen only. There are alsosignificant reasons to value TST testing over FST testing in general,many of which have been well reviewed [2]. The TST is report-edly more sensitive and more consistent in its SSRI response, anattribute that we believe is only enhanced by the head movementweighted bin sampling analysis. Furthermore, FST dose responsecurves are occasionally biphasic, which limits procedural freedomand complicates data interpretation, but this is rarely observed inthe TST. As mentioned in Section 1, the FST involves additional pro-cedural complications irrelevant to the TST such as hypothermia,water aspiration, ear tag interference or periods of limb obscura-tion during analysis. Another traditionally cited advantage of theTST is its adaptability to automated tracking [2]. As in the modi-fied FST, a behavior analysis that emphasizes qualitative differencesbetween active behaviors does not fit traditional automation meth-ods such as from a strain gauge. However, advancements in the

field of computerized behavior tracking could be adapted to theideas presented here, such as a recent video tracking method devel-oped for whisker and head movement detection in freely movingrats [48].

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A. Lockridge et al. / Behavioural

Looking forward, it will be interesting to see whether a TSTead movement weighted analysis is responsive to drugs that haveot typically been detectable in this test such as atypical antide-ressants that already show up in the FST [49,50]. In addition, themphasis of our results on 5-HT2 receptor modulation pushes theotential usefulness of this assay beyond depression research and

nto other areas where 5-HT2 receptor activity has been heavilymplicated such as obsessive behavior, bipolar disorder, serotoninyndrome toxicity, and schizophrenia [42,51–54]

cknowledgement

We would like to thank RockyLou Productions for puttingogether our video figure.

ppendix A. Supplementary data

Supplementary data associated with this article can be found, inhe online version, at http://dx.doi.org/10.1016/j.bbr.2013.02.032.

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