An investigation into behaviour, learning and memoryassessments in the juvenile Göttingen minipig treatedwith haloperidol, d-amphetamine, or scopolamine.J.C. Manton11Sequani Limited, Bromyard Road, Ledbury, Herefordshire, HR8 1LH, UK

AbstractTwenty-one naïve juvenile Göttingen minipigs were used toinvestigate techniques for behaviour, learning and memoryassessments in relation to treatment with haloperidol, d-amphetamine, and scopolamine. These substances wereselected to impair normal responses in order to assess theselectivity and sensitivity of each technique for potential use on regulatory safety evaluation studies.

An adjusted holeboard test (4 of 16 holes baited) was used toassess cognitive performance after administration of Scopolamine.Results showed that the time taken to complete the test wasincreased for animals dosed with Scopolamine when comparedwith the concurrent controls; however, there were no discernibledifferences between the Scopolamine treated animals andconcurrent controls for the number of re-visits to baited holes(Working Memory) and to unbaited holes (Reference Memory).

A 10 minute open field test was used to assess behaviouralresponse of each animal in a testing arena after administration of either haloperidol or d-amphetamine. Haloperidol and d-amphetamine produced marked changes in motor behaviour and decreased explorative behaviour, consistent with responsesdocumented in the literature. A clear distinction wasdeterminable between the behavioural profiles of thesecompounds and the concurrent controls.

In conclusion, this investigation indicated that the design of the10 minute open field test was capable of detecting behaviouralchanges in juvenile Göttingen minipigs treated with haloperidol ord-amphetamine, however, further investigations are required toassess the suitability of the adjusted holeboard test for learningand memory assessments in the juvenile Göttingen minipig treatedwith Scopolamine.

IntroductionOver the last decade, the requirement for safety evaluation ofmedicinal products in the paediatric population has intensified,thereby accelerating the need for non-clinical safetyinvestigations. The use of the minipig as a non-rodent model fornon-clinical juvenile toxicity testing is becoming increasinglypopular. In order to comply with both FDA and EU guidancedocuments released in 2006 and 2008, respectively (1, 2), thereis a need to establish methods for developmental neurotoxicityassessments in the juvenile minipig to monitor key central nervoussystem (CNS) functions, reflex ontogeny, sensorimotor function,locomotor activity, reactivity, learning and memory. In theabsence of any published data in the juvenile minipig, this studyemployed the use of two distinct tests, an adjusted holeboard test(3) to assess cognitive performance and a ten-minute open fieldtest (4) to assess behavioural response which had previously beenused in a preliminary study at Sequani (5).

The test items were selected to impair normal responses to assessthe selectivity and sensitivity of each assessment for regulatorysafety evaluation purposes. D-amphetamine sulphate wasexpected to elicit increases in activity and sensory responses,Haloperidol was expected to elicit decreases in activity, andScopolamine hydrobromide was expected to elicit decreasedlearning and memory retention. The dose levels were selectedfollowing a review of the literature (2, 6, and 7). They wereexpected to elicit the necessary pharmacological effects, butwithout marked toxicity.

Materials and MethodsThe following table summarises the groups and doses used during the study:

Table 1 Groups and dose levels

IM = Intramuscular, SC = Subcutaneous

Group 1 animals were designated Control animals and dosed with0.9 % (w/v) Sodium Chloride (saline) or 1% Tartaric acid in waterfor injection at a dose volume of 0.5 mL/kg. Group 2 animalswere designated as treated animals and were dosed withScopolamine hydrobromide for the Adjusted Holeboard Test andHaloperidol and d-amphetamine sulphate for the Open Field Testat a dose volume of 0.5 mL/kg.

Adjusted Holeboard TestDose administration was approximately 15 minutes prior to thecommencement of the neuro-behavioural test. The testing areameasured 3.2 m x 3.2 m (Figures 1 and 2) and contained 16equally-spaced buckets which were secured to the floor of thetesting arena in which visual cues were placed to aid spatialorientation. Buckets were baited under grid-perforated falsebottoms to minimise the olfactory response. For each run, 4 of the16 buckets were baited with chocolate covered raisins. Adjacentto the testing area was a holding area, where the minipigs wereheld for approximately 2 minutes prior to being tested toacclimatise.

Group

Number of animalsTestitems

Doseroute

Neuro-Behavioural

Test

Doselevel

(mg/kg)Males Females

1 6 4

0.9 % (w/v)Sodium Chloride

(saline)

SCAdjusted

Holeboard Test

-IMOpen Field

Test

1% Tartaric acidin water forinjection

SCOpen Field

Test

2 6 5

Scopolaminehydrobromide

IMAdjusted

Holeboard Test0.05

d-amphetaminesulphate

SCOpen Field

Test0.8

Haloperidol SCOpen Field

Test0.04

Figure 1 Schematic of Holeboard Test Arena

Figure 2 Minipig within theHoleboard Test Arena

To begin the run, the minipig entered the testing area via theguillotine door and the timer started when the first foot enteredthe testing arena. Each minipig had a 10 minute time limit inwhich to find all 4 chocolate raisins. Once all 4 chocolate raisinshad been found, a bell was rung, the trial stopped and the timeelapsed was recorded. If the minipig did not locate all 4 chocolateraisins in the 10 minute test duration, a bell was rung, the trialstopped and the maximum time limit was recorded as >600 seconds. At the end of each run, the minipig exited thetesting area via the swing door and returned back to its home pen.All of the buckets and the testing area were cleaned before thestart of the next run. Each run lasted a maximum of 10 minutes induration. The trials were recorded by video capture using a DigitalVideo Camera. The following data were recorded for each run:

• Start time and end time of run.

• Bucket number that the minipig investigated. This was recordedevery time the minipig puts it snout above or in a bucket.

• An error was scored whenever the minipig visited an unbaitedbucket or a bucket where it already had collected the bait.

• The bucket number that contains a chocolate raisin if this was found.

• Total time taken to find all 4 chocolate raisins.

Data AnalysisMeasures of working and reference memory were calculated perminipig and run. Working memory was defined as (number of foodrewarded visits)/(number of visits and revisits to the baited set ofholes) (4). Therefore, this measure represented the percentage ofall visits to the baited set of holes that had been positivelyreinforced with food. Reference memory was defined as (numberof visits and revisits to the baited set of holes)/(number of visitsand revisits to all holes)(4). This measure expresses the number ofvisits to the baited set of holes as a percentage of the totalnumber of visits to all the holes.

Open Field TestDose administration was approximately 30 minutes prior to thecommencement of the neuro-behavioural test. The testing arenameasured 3.2 m x 3.2 m, and rubber matting covered the floor ofthe entire test arena. The floor of the test arena was divided into12 approximately equal sections (using white tape), as depicted in Figures 3 and 4. Adjacent to the testing arena was a holdingarea, where each minipig was held for approximately 2 minutes prior to being tested to acclimatise.

Figure 3 Schematic of Open Field Arena

Each minipig was introduced into the testing arena and the timerwas started when the first foot entered the testing arena. Theminipig was observed remotely throughout the test and itsbehaviour was recorded once every 10 seconds (see Table 2 forthe appropriate behaviours). The test lasted 10 minutes induration and on completion of the test, a bell was rung, the teststopped and the minipig was removed from the testing arena andreturned back to its home pen. The floor of the arena was cleanedbetween each test run. Each run was recorded by video captureusing a Digital Video Camera.

• During each run, the start and end time, and the type ofbehaviour the minipig displayed were recorded directly.

• The following were recorded/counted retrospectively for eachrun, by reviewing the recorded test runs after test completion.

• The number of times the minipig crosses a grid-line into a newsection (defined as when the shoulder blades had crossed thegrid-line and entered a new section).

• Ambulation (the number of sections entered in the 10 minutetest duration).

• Motor slowness: Qualitative assessment of the speed of minipigmovement (Scale : 4 = fast, 3 = moderate speed, 2= slowspeed, 1= stationary).

• Behavioural shifts: the total number of behavioural changesduring the run.

Table 2 Recorded behaviours (3)

Behavioural Type Definition

Motor behaviour:

StandingStanding still for more than 2 seconds withoutexploring the surroundings

Walking Walking at least one forward step

Explorative behaviour:

ExplorationSniffing or manipulating the surroundings in a non-stereotypical manner (for more than 2 seconds)

Scanning Turning of head while looking around

Potential conflict behaviour:

Head dippingMomentary lowering and lifting of the head to the floor(for more than 2 seconds)

Intentional behaviourInitiation of a behaviour without completion of the pattern.

Comfort behaviourScratching of body with hoofs or via use of thesurroundings. Stretching of body

EliminationDeposition of urine or faecesNon-forward locomotion

Escaping:

Attempting to get out of the arena by jumping towards the wall

Potentially abnormal behaviour:

Non-forward locomotionNon-locomotory leg movements, backward locomotionor rotation around the hind legs (more than 180˚)

PosturingStanding in an awkward, odd or unphysiologicalposition (for more than 2 seconds)

Head/facial/oral activityHead shaking, jerking head movements, abnormalfacial movements and chewing that are not associatedwith exploration, licking or yawning

Distant lookStanding without focusing and with a distant look (for more than 2 seconds)

Stereotypic behaviourAny behaviour having a stereotyped appearance (i.e.behaviour is repetitive, shows little variation and withno obvious function)

Other behaviour: Different behaviour to those listed above.

Figure 4 Minipig within theOpen Field Arena

RESULTS

Adjusted Holeboard TestResults showed that the time taken to complete the test wasincreased for animals dosed with Scopolamine when comparedwith the concurrent controls as shown in Figure 5; however, therewere no discernible differences between the Scopolamine treatedanimals and concurrent controls for the number of re-visits tobaited holes (Working Memory) and to unbaited holes (ReferenceMemory) as shown in Figures 6 and 7.

Figure 5 Group Mean run times (seconds)

Figure 6 Group Mean Working Memory Scores

Figure 7 Group Mean Reference Memory Scores

Open Field TestHaloperidol and d-amphetamine produced marked changes inmotor behaviour and decreased explorative behaviour as shown inFigures 8 and 9. A clear distinction was determinable betweenthe behavioural profiles of these compounds and the concurrentcontrols. These results showed that induced changes indopaminergic neurotransmission caused distinct and quantifiablebehavioural responses in juvenile Göttingen minipigs subjected toa 10 minute open field test.

Effects of HaloperidolAdministration of Haloperidol significantly increased behaviourssuch as standing (p<0.001), scanning (p<0.01), and non-forwardlocomotion (p<0.05) when compared with the controls.Explorative behaviour was significantly decreased (p<0.01) forHaloperidol treated minipigs when compared with the controls.All other behaviours were unaffected by the administration ofHaloperidol and were similar to the concurrent controls.

Figure 8 Effects of Haloperidol on Open Field Behaviour.

Effects of d-amphetamineAdministration of d-amphetamine significantly increasedbehaviours such as standing (p<0.001) and scanning (p<0.05)when compared with the controls. Explorative behaviour wassignificantly decreased (p<0.001) for d-amphetamine treatedminipigs when compared with the controls. These recordedbehaviours complement the impression of d-amphetamine treatedminipigs having a fragmented and rigid behavioural pattern withshort bouts of walking, followed by standing alert with numerousquick turns of the head (scans) as described by Lind et al. (4). All other behaviours were unaffected by the administration of d-amphetamine and were similar to the concurrent controls.

Figure 9 Effects of d-amphetamine on Open Field Behaviour

ConclusionThis investigation indicated that the design of the 10 minute openfield test was capable of detecting behavioural changes in juvenileGöttingen minipigs treated with haloperidol or d-amphetamine,however, further investigations are required to assess thesuitability of the adjusted holeboard test for learning and memoryassessments in the juvenile Göttingen minipig treated withScopolamine.

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References1. United States Food and Drug Administration (FDA). Guidance document. Non-clinical safety evaluation of pediatric drug products. February 2006.

2. European Medicines Agency (EMEA), Committee for Human Medicinal Products(CHMP).Guideline on the need for nonclinical testing in juvenile animals ofpharmaceuticals for paediatric indications. January 2008.

3. Lind N.M., Arnfred S.M., Hemmingsen R.P., Hansen A.K., Jensen K.H. Open FieldBehaviour and Reaction to Novelty in Göttingen Minipigs: Effects of Amphetamineand Halperidol. Scand. J. Lab. Anim. Sci. No.2. 2005; 32: 103-112.

4. Arts J.W.M., van der Staay F.J., Dinand Ekkel E. Working and Reference Memory ofPigs in the Spatial Holeboard Discrimation Task. Behavioural Brain Research.2009; 205: 303-306.

5. Manton J.C. Preliminary investigation of behaviour, learning and memoryassessments in the juvenile Göttingen minipig. Reproductive Toxicology 2010;30(2): 241.

6. van der Staay F.J., Pouzet B., Mahieu M., Nordquist R.E., Schuurman T. The d-amphetamine-treated Gottingen minature pig: an animal model for assessingbehavioural effects of antipsychotics. Psycopharmacology 2009; 206: 715-729.

7. Nielsen T.R., Kornum B.R., Moustgaard A., Gade A., Lind N.M., Knudsen G.M. Anovel spatial Delayed Non-Match to Sample (DNMS) task in the Gottingen minipig.Behavioural Brain Research 2009; 196: 93-98.

AcknowledgementsThanks to the technical and scientific staff of Sequani for their participation in the

preparation of this poster.

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