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The short-term effect of Methylphenidate

on swim speed in zebrafish larvae

Dóra Dögg Kristófersdóttir

2014 BSc in Psychology

Author: Dóra Dögg Kristófersdóttir

ID number: 040183-7449

Supervisor: Jack Ernest James

Department of Psychology

School of Business

EFFECT OF METHYLPHENIDATE ON ZEBRAFISH 2

Abstract

Methylphenidate is a psychostimulant routinely prescribed for Attention Deficit Hyperactivity

Disorder (ADHD) early in life, with contradictory reports on optimal dosage sizes. Exhaustive

human research on it would be expensive and unethical, subjecting children or young adults to

random groups. Various animal studies using intravenous administrations are not applicable

to deduction on normative human use of Methylphenidate. The present study applied

zebrafish larvae, seven, ten and eleven days post-fertilization (dpf) swimming in four dosages

(zero, 4.63, 9.25 and 18.50) µmol Methylphenidate during day and night to see the change on

locomotor behavior in cm/s. The highest dose reduced locomotion compared to the other

groups during day and night, additionally there was a change between day and night with the

larvae inducing motion during night. The control, low, and medium groups were neither

different from each other nor between day and night. The highest dosing reduced locomotion

significantly, to such a degree that might imply a neurotoxic effect that neither completely

wash-out during the day nor night. The control group’s constant locomotion between day and

night might indicate the lighting malfunctioning or fasting affecting the larvae. More research

on Methylphenidate is needed.

Útdráttur

Methýlfenidat er geðörvandi lyf sem er venjubundið gefið ungu fólki með athyglisbrest með

ofvirkni (ADHD) og rannsóknir hafa almennt ekki verið sammála um skammtastærðir fyrir

hagstæðastan árangur. Ítarlegar manna-rannsóknir væru dýrar og það bryti gegn siðarreglum

að láta ungt fólk tilviljunarkennt í rannsóknarhópa. Dýrarannsóknir með lyfinu í sprautuformi

eru ekki yfirfæranlegar á eðlilega notkun manna. Umrædd rannsókn notaði sebrafiska-lirfur

sjö, tíu og ellefu dögum eftir frjóvgun og lét þær synda í fjórum skammtastærðum (núll, 4.62,

9.25 og 18.50) µmól Methýlfenidat yfir dag og nótt til að sjá breytingu á hreyfingu mældri í

cm/s. Hæðsti skammturinn dró marktækt úr hreyfingu miðað við hina hópana, ásamt því að

auka hreyfingu sína frá degi til nóttu. Það var hvorki marktækur munur á viðmiðunar hópnum,

lægsta og miðlungs skammtahópunum né milli dags og nætur. Hæsti skammtahópurinn dró

marktægt úr hreyfingu, sem gefur mögulega til kynna skaðleg áhrif á taugavirkni sem nær

hvorki að eyðast fyllilega yfir daginn né nóttina. Stanslaus hreyfing viðmiðunarhópsins frá

degi til nætur gæti gefið til kynna tækjabilun lýsingar eða að fasta lirfanna hafi haft áhrif á

hegðun þeirra. Þörf er á frekari rannsókum á efninu Methýlfenidat.


Foreword and acknowledgement

Submitted in partial fulfillment of the requirements of the BSc Psychology degree,

Reykjavík University, this thesis is presented in the style of an article for submission to a

peer-review journal.


The Short-term Effect of Methylphenidate on Swim Speed in Zebrafish Larvae

Methylphenidate and Attention Deficit Hyperactivity Disorder

The importance of adequate dosage size and administration method in animal studies

when comparing to human application is increasingly evident (Gerasimov et al., 2000). This

applies to Methylphenidate, a psychostimulant that is one of the principal active agents in

stimulants administered to attention-deficit/hyperactivity disorder (ADHD) patients of all age

groups (Faraone, Biederman, Spencer, & Aleardi, 2006; Geissler & Lesch, 2011; Kollins,

2008) as well as in other drug research. One of the most frequently prescribed medications

containing Methylphenidate is Ritalin (Volz, 2008), which is routinely prescribed for ADHD

symptoms (Kimko, Cross & Abernethy, 1999; Iversen & Iversen, 2007; Kokel & Peterson,

2008; Kollins, 2008), primarily for children and young adults for chronic use (Andersen &

Navalta, 2004).

ADHD is generally a childhood condition, although, it can last into adulthood, and

consists of behavioral, academic, and social disadvantage of inattention, disorganization,

and/or hyperactivity-impulsivity (American Psychiatric Association, 2013). Children with

ADHD make more errors of omission and commission, are more irritable, distracted, and

impulsive, have a longer response time, and perform worse than healthy controls on average

(Lecendreux & Konofal, 2000). Administration of Methylphenidate provides short-term relief

from the core symptoms of ADHD, enabling most to perform more like, if not identical to

healthy peers, (Faraone et al., 2006; Kimko et al., 1999). Stimulants such as Methylphenidate

work on neurosynaptic transmitters systems related to mobility, attention, inhibition, and

reward (Andresen, 2005)

Methylphenidate´s Effect on the Central Nervous System and Dosage

Attention, impulse control, and executive function are subserved in the prefrontal

cortex, and reward and locomotor processes are subserved by the nucleus accumbens. Both of


which contribute to ADHD symptoms when malfunctioning (Andersen, 2005). The

pharmacologic effect of Methylphenidate in the central nervous system enhances

catecholaminergic functions, either selectively through dopamine or noradrenaline, or by

effects from both of the neurotransmitters combined (Andersen, 2005; Heal & Pierce, 2006;

Levin et al., 2011; Volz, 2008), possibly causing Methylphenidate´s therapeutic efficacy as

well as the side-effects (Heal & Pierce, 2006; Kimko et al., 1999).

The average high-peak in short-term effect of Methylphenidate after oral intake varies

from one to four hours, which makes it a short-acting stimulant, and its concentration

following oral administration is strongest on average two hours after consumption (Kimko et

al., 1999). Dosage size has a parabolic function, with increased dosages optimizing short-term

relief of the symptoms, up to a certain plateau, after which the positive effects decrease with

more dosing (Andersen, 2005.; Repantis, Schlattmann, Laisney & Heuser, 2010; Kimko.,

1999; Stein et al., 2003). Ideal treatment dosage amount, however, has been debated, with

research inconsistent on dosing for optimal results (Anderson, 2005). With few well

controlled studies on the subject the inconsistency may be due to imprecise measuring

methods that can vary from subjective measures such as parent or teacher report, to varied

objective performance-tests and rating scales in diverse settings (Kimko et al., 1999).

Research conducted on healthy individuals comparing Methylphenidate with no drug has

revealed that it can improve cognitive performance short-term. Suggesting a possible overall

non-specific mechanism of arousal, improved attention, and vigilance measured in shortened

response time and decreased errors of omission (Andersen, 2005; Hermens et al., 2007;

Kimko et al., 1999). This has been debated, with inconsistent findings on exactly how

effective the drug is and what it enhances (Advokat, 2010; Repantis et al., 2010). The

cognitively enhancing effect has, however, been found in studies using various healthy

animal-models and in animals modified to present ADHD core symptoms (Andersen, 2005).


Methylphenidate is known to have negative short-term side-effects, some of the most

commonly reported being trouble sleeping and decreased appetite (Garland, Tripp & Taylor,

2010; Heal & Pierce, 2006; Kimko et al., 1999; Stein et al., 2003). Increased dosage amount

has been reported to intensify the negative side-effects (Heal & Pierce, 2006; Stein et al.,

2003). Reports contradict on the drug’s side-effects, when individuals conform to

Methylphenidate administration guidelines, both when sleep is measured, objectively and

subjectively. Some indicated beneficial sleep-efficacy (Repantis et al., 2010; Sobanski,

Schredl, Kettler & Alm, 2008) and others reduced sleep-efficacy (Galland et al., 2010). It has,

additionally, been demonstrated that recipients of placebo treatments have regularly reported

similar side-effect to Methylphenidate administration (Geissler & Lesch, 2011; Kimko et al.,

1999). With so many diverse and inconsistent reports on this subject, it is evident that further

research is needed (Barkley, McMurray, Edelbrock & Robbins, 1990; Repantis et al., 2010).

Normative Human Application of Methylphenidate

Prescribed drugs such as Methylphenidate are primarily administered orally to humans

(Heal & Pierce, 2006). The research literature on complex animal-models has largely reported

Methylphenidate intravenously applied, decreasing the predictive value for comparison except

for drug abuse (Andersen, 2005; Gerasimov et al., 2000; Heal & Pierce, 2006). Studies

consistently find evidence of reinforcing effects in intravenous Methylphenidate studies

inconsistent with results of oral administration of appropriate dosage-conforming individuals

(Andersen, 2005; Gerasimov et al., 2000; Kollins, 2008). More of the substance is digested

without reaching the brain after oral intake, with peak concentration after approximately two

hours, opposed to 15 minutes intravenously (Heal & Pierce, 2006; Gerasimov et al., 2000;

Kimko et al., 1999). This can be problematic when interpreting results for normative human

comparison (Anderson, 2005; Gerasimov et al., 2000). Animal studies that either use smaller

dosage sizes or administered orally have not found the same reinforcing effect (Gerasimov et


al., 2000; Koda, Ago, Cong, Takuma & Matsuda, 2010; Kollins, 2008) possibly as shorter

time from stimulus to response supplies more behaviour change (Tanno, Silberberg &

Sakagami, 2012).

Why Should Methylphenidate be Studied Further?

Defining long-term effects of Methylphenidate is problematic, and there is more

opportunity for confounding by extraneous variables (Kimko et al., 1999). Randomized

controlled long-term drug experiments on children are close to impossible to accomplish,

being both impractical and unethical to randomly assign children to a treatment plan and

sustain it over time (Vitiello, 2001). Animal and human research reveals possible long-term

effects, both in altering the brain and consequently behaviour (Andersen, 2005). Some

relating young patients with ADHD on stimulants with lower grade-retention rates and

comorbid psychological disorders subsequently in adulthood, both known risk-factors of

ADHD (Biederman, Monuteaux, Spencer, Wilens & Faraone, 2009). Others report that

ADHD, as a risk-factor for substance abuse, is less likely to lead to drug addiction when the

individuals are treated with Methylphenidate in childhood (Kollins, 2008). ADHD diagnosed

individuals treated with Methylphenidate in childhood have reported better self-esteem and

social skills later in life and in hindsight appraised their childhood more favorably than non-

treated individuals (Kimko et al., 1999).

Other research has revealed potential negative long-term effects of stimulants such as

Methylphenidate. Levin et al. (2011) indicated long-term dangers of early-life

Methylphenidate exposure on zebrafish larvae. They exposed zebrafish embryos 0-5 days post

fertilization to Methylphenidate doses that resulted in persistently altered behaviour in their

early adulthood after early developmental exposure. Their research and others directed at such

early exposure raise concern for women at childbearing age who use the drug when

conceiving and into their pregnancy as well as for the children prescribed Methylphenidate as


young as two years of age (Andersen & Navalta, 2004). The long-term effects are reported in

various animal studies across species, although, it is most consistent after stronger dosing than

applies to normative human use (Gerasimov et al., 2000; Vitiello, 2001). As stated previously,

another possible confounder may arise when the drug is administrated intravenously

(Gerasimov et al., 2000). It raises concern as research findings are used for over-

generalization purposes when the difference of the Methylphenidate concentration that

reaches the brain after oral opposed to intravenous application is undebated. While in practice

human intravenous administration of Methylphenidate is rare (Anderson, 2005; Gerasimov et

al., 2000; Grund, Lehmann, Bock, Rothenberger & Teuchert-Noodt, 2006; Vitello, 2001) with

one study reporting a level of 2% occurrence rate (Barrett, Darredeau, Bordy, & Pihl, 2005).

Despite minimal knowledge of Methylphenidates long-term effects (Kimko et al.,

1999) both positive and negative long-term effects may derive from the fact that individuals

are most likely to be chronically exposed in childhood and early adulthood (Andersen &

Navalta, 2004; Andersen, 2005). It is likely this stems from early- and mid-childhood being a

time where the dynamic brain develops rapidly with excessive production of synapses and

receptors (Andersen, 2005; Vitiello, 2001).

How to Study Methylphenidate Further?

Dosage size and normative administration of how human use of the drug must be

emulated when using non-humans as research models for direct human comparative relevance

(Andersen, 2005; Vitiello, 2001). Increased dosage size is reported to intensify the side-

effects (Stein et al., 2003) and researches are inconsistent on ideal dosage size, adding to the

importance of further study on the subject (Kimko et al., 1999). Long-term human research is

expensive and often impractical, especially if meant to analyse the impact over a lifetime, at

risk for various biases and unethical to conduct randomly and experimentally (Vitiello, 2001).

To find long-term biological and environmental consequences of Methylphenidate on


cognitive, genetic, molecular, and neural factors, preliminary studies on animals with a short

lifespan and cell models are essential (Andersen & Navalta, 2004; Geissler & Lesch, 2011).

After thorough animal research, long-term systematic experimental human research could

become a viable choice.

Zebrafish as an Animal Model for Human Comparison

Zebrafish have various benefits over other research animals making them suitable

simple vertebrate animal-models for human comparison (Hendricks, Sehgal & Pack, 2000).

Their early-life development is visible as they are see-through during their first days of life,

they are cheap in upkeep, easy to handle, procreate, and maintain. They are vertebrates with

relatively simple nervous system retaining fundamental circuits found in humans and have

been found repeatedly to have similar diurnal rhythm to humans (Appelbaum et al., 2009 ;

Carlson, 2010; Chiu & Prober, 2013; Emran & Dowling, 2010; Elbaz, Foulkes, Gothilf &

Appelbaum, 2013; Emran, Rihel, Adolph & Dowling, 2009; Hendricks, Sehgal & Pack. 2000;

Kishi, Slack, Uchiyama & Zhdanova, 2009; Saszik & Bilotta, 2001; Zhdanova, Wang,

Leclair & Danilova, 2001; Zimmerman, Naidoo, Raizen, & Pack, 2008). Sanchez and

Sanchez-Vazquez (2009) found contrary results on adult zebrafish circadian rhythm. They

revealed that in constant light; when fasting, or when free to receive food at own time-

preference in a self-feeding system, a flat locomotor activity occurred with the fish moving

congruently during the 24 hours.

Normal zebrafish lifespan in the wild is not known (Lawrence, 2007). In experimental

settings zebrafish have a parabolic lifespan consisting of initial extensive death rate, followed

by a higher survival rate, mean life expectancy of 42 month, after which death rate increases

until the maximum of 66 months (Gerhard et al., 2002). A useful way to study zebrafish is

measuring locomotion (Hurd, Debruyne, Straume & Cahyll, 1998; Saili et al., 2012) which

was applied in the present study, with swim speed in cm/s.


Aim of this study

The aim of this study was to record, measure, and analyse zebrafish larvae swim speed

cm/s during day and night for four separate dosages of Methylphenidate; zero (control), 4.63

(low), 9.25 (medium) and 18.50 (high) µmol of Methylphenidate. Particularly, the study

examined short-term effects of Methylphenidate on zebrafish larvae in order to identify the

suitable dosing amount.

Method

Participants

The subjects of this study were 604 Danio rerio zebrafish larvae where the University

of Oregon, Zebrafish International Resource Centre, provided the original fish of a wild stock

for breeding. The sample size was determined as the fish is known to have a naturally high

death rate over their first weeks and the experimental group’s early Methylphenidate exposure

likely to result in an increased death rate. Five out of eight recordings were successful, one

had 80 larvae that occupied the wells, three had 79 and one had 38 during recording (n=355).

Larvae at seven, ten and eleven- days post fertilization (dpf) were recorded successfully and

after exclusion, 324 (control n=58, low n=89, medium n=89, and high n=88) were used for

analyses.

Exclusion Criteria

If a larva was not tracked for minimum of 95% of the timeframe, it was excluded from

the statistical processing, as well as after various technical or procedural problems that could

affect and corrupt the recording of the larvae (n=280).

Statistical Analysis

The independent variables were two, the amount of drug and the phase. The mean

swim speed was assessed by two-way analyses of variance. With four levels of drug

manipulation, control (no drug), low Methylphenidate dose, medium Methylphenidate dose


and high Methylphenidate dose. There were two levels of the phase, measured in the

timeframe of light on (day) and light off (night). The dependent variable was the mean swim

speed in m/sec. The drug was a between-group comparison and the phase was a within-

subject comparison. All post hoc comparisons were subjected to Bonferroni corrections.

Significance threshold was set at p < .05 and T-tests were applied for comparison for each

condition day versus night.

Instruments

When the eggs were harvested Methylene blue was added to the tank containing the

eggs to minimize infection. After hatching the larvae were fed 0.25g larval feed per day,

every morning prior to the recording, and to remove the Methylene blue a 10 L multi-tank

constant flow system (Aquatic Habitats, Apopka, FL, USA) was used the day before

recording. During the recording larvae were not provided with feed, and the control group

only received water from the breeding tank. The three experimental groups, additionally,

received Methylphenidate, an active drug administered in three separate doses, zero (control),

4.63 (low), 9.25 (medium) and 18.50 (high) µmol of Methylphenidate to swim in. The larval

behavior, swim speed, was recorded and tracked by Ethovision version 7.0 (Noldus

Information Technology) at two dimensions with a Sony XC-E150 infrared camera (Sony) at

8.33 Hz with a 50-mm CCTV Pentax lens (Pentax). Microwell plate, with the ability to

accommodate 96 larvae in separate wells (.4cm inner dimension and .38ml solution), was held

by a custom-built transparent Plexiglass container with a constant water flow, to keep the

larvae at 27.9°C to 28.5°C. The Plexiglas container was placed under the monitor tracing the

larvae activity (Noldus Information Technology). Illumination under the container was 2551x

during the white-light phase (day) and 0lx during the infrared-dark phase (night) throughout

the recording.


Procedure

The new research stock was bred, the morning after the eggs were separated from the

adult fish and harvested, with Methylene blue added to the tank containing the eggs. When

hatched, the larvae were fed once daily with .025g larval food and 24h prior to recording a

constant 28.5°C water flow of filtered water, 10% volume per day, was added to the tank in a

10 L multi-tank constant flow system. Figure 1. reveals the procedure step by step.

Figure 1. Research Procedures

Figure 1. dpf = days post-fertilization.

Prior to each recording, solutions were measured and distributed per well one solution at a

time. Other concentrations were kept separate until it had been confirmed that a correct

concentration had been administered to the appropriate well to maintain secure separation

between drug conditions. The larvae were randomly selected from the stock, manually with

separate pipettes, and assigned a condition by a single researcher, with a fixed systematic

Breeding

new stock

• The adult fish randomly picked, between the time 20 - 21, from breeding-stock for breeding in a breeding-tank

Harvesting

• The eggs were harvested between 8 - 10 in the morning after, and methylene blue was added to the tank

Removing

methyline blue

• The previous day before the recording, filtered water flow was added to the breeding-tank to remove the methylene blue

Prepering recording

• Between 10:00 - 11:30 on the day of the recording, 80 wells of Microwell plates, filled with .38ml of each solution and larvae seven, ten and eleven dpf added to each of them

Recording

• Between 12:00 - 13:34 the recording was started and stopped 24 hours later, thereafter each recording was cut to same timeframe of 13:34 until 8:00 the morgning after


procedure for every recording of zebrafish swim speed in cm/s. First the high

Methylphenidate group was added to the wells, then the medium Methylphenidate group, next

the low Methylphenidate group and last the control group. Larvae were randomly assigned to

the groups seven, ten and eleven dpf. The microarray had 96 wells, however, technical

problems only allowed 80 wells per array to be tracked per recording.

The five separate successful recordings started at midday and ended at midday the

morning after. Three recordings were not usable because of system failure. The main lighting

in the experimental environment was twofold, white light-phase from the start of recordings

midday, until 22:00 when infrared dark-phase was turned on and visual lights were turned off.

Recordings ended at midday, after which the recorded period was cut so that all the

recordings were at the same real-time.

Results

The results showed valid recordings from 324 zebrafish larvae consisting of control

group with 58 zebrafish larvae, low Methylphenidate of 89, medium Methylphenidate of 89

and high Methylphenidate of 88. Table 1 is an ANOVA summary table that shows the main

and interaction effects between the four groups (N = 324). The repeated measures factor

Phase, of day and night, was subjected to Greenhouse-Geisser correction. There was a

significant main effect for condition, a significant main effect for Phase, and a significant

Conditions x Phase interaction effect.


Table 1.

Summary of Two-way Repeated-measures ANOVA.

Source df Mean square F

CONDIT1 (between groups) 3 2.714 16.462*

PHASE2 (within subjects) 1 0.003 22.777*

CONDIT x PHASE 3 0.001 8.138*

Error 320

Note. 1CONDIT - four experimental conditions involving swimming in the solution of 0

(control), 4.63 (low), 9.25 (medium) and 18.50 (high) µmol Methylphenidate.

2PHASE – lights on (DAY) and lights off (NIGHT).

* p < .001

In view of the significant main effect for group, post hoc tests were conducted with

Bonferroni correction. Testing the differences for the main effect between groups revealed

that the high Methylphenidate group differed from the other groups while the control, low

Methylphenidate and medium Methylphenidate groups were not statistically different. This

interaction can be seen in Figure 2. The cut-off for significance after Bonferroni correction

was .0125. T-tests were conducted to compare mean swim speed during day and night for

each group. The control group t(57) = 0.001,and the low Methylphenidate group t(88) =

0.984, were not significant, the medium Methylphenidate group t(88) = 2.415, p =.018, was

just below significance of .0125 after the correction, and the high Methylphenidate group was

significant at t(87) = 9.875, p < .001. The medium and high Methylphenidate dosed groups


were still slower than the control group during the night, although, all of the drugged groups

moved faster during the night than the day.

Figure 2. The Phase Day and Night for each Group

Figure 2. Shows larvae mean swim speed cm/s for each condition during the Day and Night.

MPH = Methylphenidate.

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Swim

sp

ead

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Control Group

Low MPH Group

Medium MPH Group

High MPH Group


Figure 3. Larvae Mean Swim Speed cm/s During the Day

Figure 3. Mean swim speed cm/s for each five-minute epoch. MPH = Methylphenidate.

Figure 3 shows recordings of larvae mean swim speed cm/s for each five-minute

epoch 13:40 until 21:20 for each condition. T-tests, as reported above, showed the three

groups, control, low Methylphenidate and medium Methylphenidate were not statistically

different from each other, while the larvae in the high Methylphenidate group moved less

during the timeframe.

The phase day with mean swim speed and standard deviation can be seen in Figure 4.

with the four conditions valid recordings, high Methylphenidate dose groups swim speed

lower than the other groups when looking at total mean swim speed during day.

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Larvae Velocity at Day

Control Low MPH dose Medium MPH dose High MPH dose


Figure 4. Mean Swim Speed during Day

Figure 4. Mean swim speed cm/s of the whole phase (day). MPH = Methylphenidate

Control group had the Mean (M) of 0,0693 and SD = 0,020, low Methylphenidate dosed

group had M = 0,0678 and SD = 0,0194, medium Methylphenidate dosed group had M =

0,0652 and SD = 0,0163 and the high Methylphenidate dosed group had M = 0,0524 and the

SD = 0,0122. The high Methylphenidate dosed group differed significantly from the control

group and the low and medium Methylphenidate groups.

Figure 5. reveals the locomotion during night with recordings for each condition

during five-minute bins from 22:20. until 8:00 where larvae behaviour change mean swim

speed was used for analysis.

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Control Group Low MPH Group Medium MPH Group High MPH Group

Mea

n S

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Sp

ead

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/s

Larvae Mean Swim Spead Day


Figure 5. Mean Swim Speed cm/s at Night

Figure 5. Larvae mean swim speed every five minutes in cm/s in constant phase night from

22:20 until 08:00. MPH = Methylphenidate.

Figure 6 shows the mean swim speed at night during the recording with standard

deviation (SD). Control group had the M of 0,0693 and SD = 0,0158, low Methylphenidate

group had M = 0,0697 and SD = 0,0085, medium Methylphenidate group had M = 0,0689 and

SD = 0,0084 and the high Methylphenidate group had M = 0,0640 and the SD = 0,0090

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Larvae Swim Speed at Night

Control Low MPH dose Medium MPH dose High MPH dose


Figure 6. The Swim Speed of Zebrafish Larvae during Night

Figure 6. shows the mean swim speed during night with Standard Deviation bars.

MPH=Methylphenidate

Discussion

The purpose of the study was to analyse zebrafish larvae mean swim speed for four

dosages, zero, 4.62, 9.25 og 18.50 µmol of Methylphenidate to identify suitable dosing for

research on the species with this drug. The independent variables were two, phase and

condition. The phase had two stages, day and night, and the condition had four stages of

Methylphenidate exposure, low, medium and high. The dependent variable was the mean

swim speed cm/s during their first day and night of Methylphenidate exposure.

The larvae in the high Methylphenidate group moved slower than the other groups, so

slow that it could be interpreted as a status similar to an overdose or a toxic effect. The

control, low, and medium Methylphenidate groups were similar to each other both during day

and night, with no statistical difference. The high Methylphenidate group, however, showed

lack of movement compared to the other groups, both during day and night, with a slight rise

in movement during the night after being in the solution for the whole day, possibly as the

0

0,01

0,02

0,03

0,04

0,05

0,06

0,07

0,08

0,09

Control Group Low MPH Group Medium MPH Group High MPH Group

Mea

n S

wim

Sp

ead

cm

/s

Larvae Swim Spead at Night


drug in the solution was metabolized by the larvae. Looking at movement during the phases

for different conditions, their mean swim speed showed some tendency for bigger

Methylphenidate dosage to add to the increased motion. The high dosed group with high

statistical difference and the marginal effect for the medium dosed group and the low dosed

group closer to the control group. The larvae moved faster during the following night than the

previous day. The report by Galland et al. (2010) where Methylphenidate given during the

day deduces sleep efficiency during night might be supported by these findings. The high

Methylphenidate group, however, had the highest increase in motion from day to night and

was still not as fast at night as any of the other groups during the previous day, perhaps as the

high Methylphenidate group daytime swim speed was so low. It could indicate that the larvae

needed time to recuperate from toxic effects of the high Methylphenidate dose and that the

recovery was not complete.

The Levin et al (2011) research group studied zebrafish larvae with Methylphenidate

dosages about 26% stronger per group than the present study, given during the first five dpf

and examined three months subsequently. They found that the long-term effect was only

visible for the high Methylphenidate dosed group, perhaps indicating that the large dose is

neurotoxic. Their deduction was that humans should refrain from taking the drug while

reproducing. A valid question would then be: How much does result of a neurotoxic dose on

zebrafish larvae apply to normative research on human (regarding prescription and

application) were the present research finds that the 26% more diluted dose reviles such

reduction in locomotion.

The present research studied the larvae in the first day and night of Methylphenidate

exposure. The control group and the two Methylphenidate groups receiving the smaller

dosages were more like one group, with no statistical difference between them. It was

surprising that the control group had no statistical difference where the larvae move less


during the night when most other research suggest the zebrafish are a diurnal species (Elbaz et

al., 2013; Hurd et al., 1998). As the control group in the precent study did not respond as most

normal zebrafish larvae have been recorded to behave in the past, this research still needs to

be repeated and improved in order to clarify whether technical failures were responsible for

current results. The most plausible explanation would be that the lighting in this research was

not set correctly, as the various lighting in zebrafish environment has been proven to affect

their diurnal rhythm (Padilla, Hunter, Padnos, Frady, and MacPhail, 2011). The previous

research findings on zebrafish larvae is parallel to the study of Sanchez and Sanchez-Vazquez

(2009) on adult zebrafish where when fasting and in constant light the fish sustained their

locomotor activity sequence during the 24 hours. The locomotion was different when the fish

was fed and if the regular feeding time was at night the fish did not move as much as if

feeding was during day. Down to a cellular level, Cahill (2002) indicated that the zebrafish

circadian rhythm might derive from light-sensitive oscillators present in the peripheral tissues

with pacemakers distributed over the organism and entrained by light, independently of other

factors. Other research simply indicates zebrafish loose vision at night (Emran and Dowling,

2010; Emran et al., 2009) which could explain why most research have found that zebrafish

have similar diurnal rhythm to humans.

Additional explanations to the present research findings could be found in recent

research that has come out since the present research was conducted and points to critical

importance of the width and depth of the wells, and the post-fertilization age of the zebrafish

larvae, as vital influences on zebrafish larvae locomotor behaviour (Ingebretson and Masino,

2013; Padilla et al., 2011). Considering recent research, it would be informative to repeat the

present study to remove all doubt about lighting mechanisms malfunctioning and to study

further the relationship and effects of diverse lighting on zebrafish behaviour, especially

regarding diurnal rhythms when in constant lighting conditions. Since the present research


was conducted, Saili et al. (2012) have been able to alter zebrafish so that they revile ADHD

symptoms, which if applied to the present research, could improve it even further.

Furthermore, it might be informative to examine how gender is influenced by these

conditions, as the present study did not consider gender specifically.

Methylphenidate as a helpful solution to fundamental ADHD dysfunction is evident

but the lack of consistent research on the drug is a weakness that needs to be remedied.

Dosages, how and when to apply them, are all worthy questions when research continues to

be inconsistent. The present research showed indications that previous studies may not have

dosed equivalently to normative human application as short-term effect on humans after

normative oral administration has not been reported to be drastically reduced locomotion.

There have, however, been reports of more side-effects following higher dosing. If the short-

term effect of the substance on zebrafish larvae is to be applicable to studies on humans, the

dosage levels applied for further studies must fit the short-term effects on humans. If this

cannot be obtained, then applying zebrafish larvae as animal models for short- and long-term

effects of the drug might not be the best model. The question of how to apply animal drug

studies to deduction of human comparison is still left unanswered. If drug research on animals

is to be ethically conducted then results should further practical knowledge.


References

Advokat, C. (2010). What are the cognitive effects of stimulant medications? Emphasis on

adults with attention-deficit/hyperactivity disorder (ADHD). Neuroscience and

Biobehavioral Reviews, 34(8), 1256-1266. doi:10.1016-j.neubiorev.2010.03.006

American Psychiatric Association. (2013). Diagnostic and statistical manual of mental

disorders (5th

ed.). Arlington, VA: American Psychiatric Publishing.

Andersen, S. L., & Navalta, C. P. (2004). Altering the course of neurodevelopment: a

framework for understanding the enduring effects of psychotropic drugs. International

Journal of Developmental Neuroscience, 22(5-6),423-440.

doi.org/10.1016/j.ijdevneu.2004.06.002

Appelbaum, L., Wang, G., X, Maro, G. S., Mori, R., Tovin, A., Marin, W., Yokogawa, T.,

Kawakami, K., Smith, S. J., Gothilf, Y., Mignot, E., & Mourrain, P. (2009). Sleep-

wake regulation and hypocretin-melatonin interaction in zebrafish. PNAS, 106(51),

21942-21947. doi:10.1073/pnas.906637106

Barkley, R, A. McMurray, M, B. Edelbrock, C, S., & Robbins, K. (1990). Side effects of

methylphenidate in children with attention deficit hyperactivity disorder: a systematic,

placebo-controlled evaluation. Pediatrics, 86(2), 184-192. Retrieved from

http://web.ebscohost.com/ehost/pdfviewer/pdfviewer?sid=61654f0b-7b07-4536-9749-

2274524ab636%40sessionmgr114&vid=2&hid=122

Barrett, S. P., Darredeau, C., Bordy, L. E., & Pihl, R. O. (2005). Characteristics of

methylphenidate misuse in a university student sample. The Canadian Journal of

Psychiatry, 50(8), 457-461. Retrieved from

http://search.proquest.com/docview/222806521?accountid=28419

Biederman, J., Monuteaux, M. C., Spencer, T., Wilens, T. E., & Faraone, S. V. (2009). Do

stimulants have a protective effect on the development of psychiatric disorders in


youth with ADHD? A ten-year follow-up study. Pediatrics, 124(1), 71-78. doi:

10.1542/peds.2008-3347.

Cahill, G. M. (2002). Clock mechanisms in zebrafish, Cell Tissue Research. 309(1), 27-34.

doi 10.1007/s00441-002-0570-7

Chiu, C. N., & Prober, D. A. (2013). Regulation of zebrafish sleeps and arousal states: current

and prospective approaches. Frontiers in Neural Circuits, 7(58), 1-14.

doi:10.3389/fncir.2013.00058

Elbaz, I., Foulkes, N. S., Gothilf, Y., & Appelbaum, L. (2013). Circadian clocks, rhythmic

synaptic plasticity and the sleep-wake cycle in zebrafish. Frontiers in Neural Circuits,

7(9). doi: 10.3389/fncir.2013.00009

Emran, F., & Dowling, J. E. (2010). Larval zebrafish turn off their photoreceptors at night.

Communicative & integrative biology. 3(5), 430-432. doi:10.4161/cib.3.5.12158

Emran, F., Rihel, J., Adolph, A. R., & Dowling, J. E. (2009). Zebrafish larvae lose vision at

night. PNAS. 107(13), 6034-6039. doi: 10.1073/pnas.0914718107

Faraone, S. V., Biederman, J., Spencer, T. J & Aleardi, M. (2006). Comparing the efficacy of

medications for ADHD using meta-analysis. Medscape General Medicine, 8(4), 1-16.

Retrieved from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1868385/

Galland, B. C., Tripp, E. G., & Taylor, B. J. (2010). The sleep of children with attention

deficit hyperactivity disorder on and off methylphenidate: a matched case-control

study. Journal of Sleep Research, 19 (2),366-373. doi: 10.1111/j.1365-

2869.2009.00795.x

Geissler, J., & Lesch, K. P. (2011). A lifetime og attention-deficit/hyperactivity disorder:

diagnostic challenges, treatment and neurobiological mechanisms. Expert Review of

Neurotherapeutics, 11(10), 1467-1484. doi: 10.1586/ern.11.136


Gerasimov, M. R., Franceschi, M., Volkow, N. D., Gifford, A., Gatley, S. J., & Marsteller, D.

(2000). Comparison between intraperitoneal and oral methylphenidate administration:

a microdialysis and locomotor activity study. The Journal of Pharmacology and

Experimental Therapeutic, 295(1), 51-57. Retrieved from

http://jpet.aspetjournals.org/content/295/1/51.full.pdf+html

Gerhard, G. S., Kauffman, E. J., Wang, X., Stewart, R., Moore, J. L., Kasales, C. J., &

Demidenko, E. (2002). Life spans and senescent phenotypes in two strains of zebrafish

(Danio rerio). Experimental Gerontology, 37(8-9), 1055-1068. doi.org/10.1016/S0531-

5565(02)00088-8

Heal, D. J., & Pierce, D. M. (2006). Methylphenidate and its isomers. Their role in the

treatment of attention-deficit hyperactivity disorder using a transdermal delivery

system. CNS Drugs, 20(9), 713-738. doi:10.2165/00023210-200620090-00002

Hendricks, J. C., Sehgal, A., & Pack, A. I. (2000). The need for simple animal model to

understand sleep. Progress in Neurobiology, 61(4), 339-351. doi.org/10.1016/S0301-

0082(99)00048-9

Hermens, D. F., Cooper, N. J., Clark, C. R., Debrota, D., Clarke, S. D,. & Williams L. M.

(2007). An integrative approach to determine the best behavioral and biological

markers of methylphenidate. Journal of Integrative Neuroscience, 6(1), 105-140. doi:

10.1142/S0219635207001441

Hurd, M. W., Debruyne, J., Straume, M., & Cahyll, G. M. (1998). Circadian rhythms of

locomotor activity in zebrafish. Physiology & Behavior, 65(3), 465–472.

doi.org/10.1016/S0031-9384(98)00183-8

Ingebretson, J. J., & Masino, M. A. (2013). Quantification of locomotor activity in larval

zebrafish: considerations for the design of high-throughput behavioral studies.

Frontiers in Neural Circuits, 7(109), 1-9. doi: 10.3389/fncir.2013.00109


Kimko, H. C., Cross, J. T., & Abernethy, D. R. (1999). Pharmacokinetics and clinical

effectiveness of Methylphenidate. Clinical Pharmacokinetics, 37(6), 457-470.

doi:10.2165/00003088-199937060-00002

Kishi, S., Slack, B. E., Uchiyama, J., & Zhdanova, I. V. (2009). Zebrafish as a genetic model

in biological and behavioral gerontology: Where development meets aging in

vertebrates- a mini–review. Gerontology, 55(4), 430-441. doi: 10.1159/000228892

Koda, K., Ago, Y., Cong, Y., Kita, Y., Takuma, K., & Matsuda, T. (2010). Effects of acute

and chronic administration of atomoxetine and methylphenidate on extracellular levels

of noradrenaline, dopamine and serotonin in the prefrontal cortex and striatum of

mice. Journal of Neurochemistry, 114(1), 259-270. doi:10.1111/j.1471-

4159.2010.06750.x

Kokel, D., & Peterson, R. T. (2008). Chemobeahavioural phenomics and behaviour-based

psychiatric drug discovery in the zebrafish. Briefings in Functional Genomics and

Proteomics, 7(6), 483-490. doi:10.1093/bfgp/eln040

Kollins, S. H. (2008). A qualitative review of issues arising in the use of psychostimulant

medications in patients with ADHD and co-morbid substance use disorders. Current

Medical Research and Opinion, 24(5), 1345-1357. doi:10.1185/030079908x280707

Lawrence, C. (2007). The husbandry of zebrafish (Danio rerio): a review. Aquaculture, 269(1-

4), 1-20. doi: 10.1016/j.aquaculture.2007.04.077

Levin, E. D., Sledge, D., Roach, S., Petro, A., Donerly., & Linney, E. (2011). Persistent

behavioral impairment caused by embryonic methylphenidate exposure in zebrafish.

Neurotoxicology and Teratology, 33(6), 668-673. doi: 10.1016/j.ntt.2011.06.004

Padilla, S., Hunter, D. L., Padnos, B., Frady, S., & MacPhail, R, C. (2011). Assessing

locomotor activity in larval zebrafish: Influence of extrinsic and intrinsic variables.

Neurotoxicology and Teratology, 33, 624–630. doi.org/10.1016/j.ntt.2011.08.005


Repantis, D., Schlattmann, P., Laisney, O., & Heuser, I. (2010). Modifinil and

methyphenidate for neuroenhancement in healthy individuals: a systematic review.

Pharmacological Research, 62(3), 187-206.

Saili, K. S., Corvi, M. M., Weber, D. N., Patel, A. U., Das, S. R., Przybyla, J., … Tanguay, R.

L. (2012). Neurodevelopmental low-dose bisphenol A exposure leads to early life-

stage hyperactivity and learning deficits in adult zebrafish. Toxicology, 291(1-3), 83-

92. doi: 10.1016/j.tox.2011.11.001

Sanchez, J, A., & Sanchez-Vazquez, F, J. (2009. Feeding entrainment of daily locomotor

activity and clock gene expression in zebrafish brain. Chronobiology International,

26(6), 1120-1135. doi: 10.1080/07420520903232092

Saszik, S., & Bilotta, J. (2001). Constant dark-rearing effects on visual adaptation of the

zebrafish ERG. International Journal of Developmental Neuroscience, 19(7), 611-619.

doi.org/10.1016/S0736-5748(01)00051-X

Sobanski, E.,M.D., Schredl, M., phD., Kettler, N., M.D., & Alm, B., M.D. (2008). Sleep in

adults with attention deficit hyperactivity disorder (ADHD) before and during

treatment with methylphenidate: a controlled polysomnographic study. Sleep, 31(3),

375-381. Retrieved from http://europepmc.org/articles/PMC2276739?pdf=render

Stein, M. A., Sarampote, C. S., Waldman, I. D., Robb, A. S., Conlon, C., Pearl, P. L., …

Newcorn, J. H. (2003). A dose-response study of oros methylpenidate in children with

attention-deficit/hyperactivity disorder. Pediatrics, 112 (5). doi:

10.1542/peds.112.5.e404

Tanno, T., Silberberg, A., & Sakagami, T. (2012). Discrimination of variable schedules is

controlled by interresponse times proximal to reinforcement. Journal of the

Experimental Analysis of Behaviour, 98(3), 341-354. doi: 10.1901/jeab.2012.98-341


Vitiello, B. (2001). Long-term effects of stimulant medications on the brain: Possible

relevance to the treatment of attention deficit hyperactivity disorder. Journal of Child

and Adolescent Psychopharmacology, 11(1), 25-34.

doi.org/10.1089/104454601750143384

Volz, T. J. (2008). Neuropharmacological mechanisms underlying the neuroprotective effects

of methylphenidate. Current Neuropharmacology, 6(4), 379-385. doi:

10.2174/157015908787386041

Zhdanova, I. V., Wang, S. Y., Leclair, O. U., & Danilova, N. P. (2001). Melatonin promotes

sleep-like state in zebrafish. Brain Research, 903(1-2), 263-268.

doi.org/10.1016/S0006-8993(01)02444-1

Zimmerman, J. E., Naidoo, N., Raizen, D. M., & Pack, A. I. (2008). Conservation of sleep:

insights from non-mammalian model systems. Trends in Neurosciences, 31(7), 371-

376. doi:10.1016/j.tins.2008.05.001

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