Tamiqua Graham

Senior Biology Research Composition

Professor Marguerite Kenney
May 2, 200
Investigating the Effects of Caffeine Consumption on Learning and Memory in
Planarians (Dugesia trigina): A Model for Comparative Study of uman !eings
Introduction
Caffeine is the common name for , !, "#tri methyl $anthine% &hen
purifi e', caffeine pro'uces an intensel y (itt er )hite po)'er that pro*i'es
a 'istinct i*e tast e in soft 'rin+s% The )or' ,caffeine, came from the
German )or' +aff ee an' the -rench )or' caf , each meaning coffee% .fter
ingest ing caffeine, i t is compl etel y a(sor(e' )ithin !0 to /0 minutes, an' its
effects su(stantially 'iminish )ithin a(out three hours% 1t is e*entually e$crete' so there
is no accumulation in the (o'y% Caffeine has (een sho)n to affect moo', stamina, the
cere(ral *ascular system, an' gastric an' colonic acti*ity% 1t is also a stimulant, )hich,
upon entering the (loo'stream, causes the capillaries to 'ilate, thus increasing (loo' flo)%
This increase' (loo' flo) allo)s most (o'ily acti*ities to perform an' happen faster% 1t is
+no)n as the most )i'ely use' 2psychoacti*e3 'rug in the )orl'%

Planarian, the common name for se*eral genera of the free #li*ing 4tur(ellarian5
flat)orms (elonging to the or'er Tricla'i'a, a name that 'eri*es from their characteristic
three#(ranche' 'igesti*e ca*ities% Most species range from 67 in% to a(out in% in length
4%!2#2%0/ cm5 although some giant tropical forms range up to 2 ft% 480 cm5% The 'ifferent
1
http://www.medicinenet.com/cafeine/article.htm
species are )hite, gray, (ro)n, or (lac+9 a fe) forms are transparent% Many are stripe' or
strea+e' an' some of the large terrestrial species are (rightly colore'% .lthough
planarians can (e foun' in marine or moist terrestrial ha(itats, most inha(it fresh)ater
areas% They cra)l a(out o*er a trail of mucus that is secrete' (y speciali:e' epi'ermal
cells9 the smaller forms mo*e a(out (y means of cilia on their *entral, or lo)er, surface,
an' larger species utili:e muscular contractions as )ell% Tactile an' chemorecepti*e cells,
locate' in the epi'ermis, ser*e as general sense organs% 1n many species these cells are
clumpe' in lo(es at the si'es of the hea'% Most planarians are also light sensiti*e an' in
some, pigmente' light#sensiti*e cells are clumpe' in t)o cups that ser*e as primiti*e
eyes% Planarians are usually either carni*orous or sca*engers% The mouth is locate' near
the mi''le of the *entral surface% The tu(e li+e pharyn$ can (e a*erte' from the mouth
an' inserte' into the prey9 foo' is partially 'igeste' e$ternally (efore it is suc+e' into the
gut% Planarians are hermaphro'itic9 each in'i*i'ual )orm contains (oth male an' female
organs, an', most commonly, they repro'uce se$ually% ;o)e*er, species similar to the
62#in%#long 4%2"#cm5 Dugesia tigrina, the most common planarian in the <nite'
States, are much stu'ie' in classrooms an' la(oratories for their a''itional capacity to
repro'uce ase$ually (y trans*erse rupture of the (o'y= a rupture line 'e*elops (ehin' the
mouth, an' )hile the (ac+ half of the )orm is anchore', the front half mo*es for)ar'
until the )orm snaps in half% >ach half regenerates the missing parts% Such planarians can
also regenerate parts that are cut from the (o'y% Planarians are classifie' in the phylum
Platyhelminthes, class Tur(ellaria, or'er Tricla'i'a%
2
The planarian ner*ous system comprises of the cere(ral ganglia, a cluster of ner*e
cells equi*alent to a (rain, longitu'inal ner*e cor's an' peripheral ner*e ple$uses, as
2
http://www.encyclopedia.com/topic/planarian.aspx
sho)n in Fig. 1 (elo)% The central ner*ous system of the planarian is sai' to (e the
starting point for the e*olution of the *erte(rate central ner*ous system% 1n fact, neurons
of the planarian (rain share more li+eness )ith those of *erte(rate (rain than they 'o )ith
those of the a'*ance' in*erte(rates9 hence the planarian is propose' to (e the ancestor of
the *erte(rate (rain%
!

"igure # Encyclopedia !rittanica$ Inc (%&&%)
1n or'er to un'erstan' the arousing effect of caffeine in the (rain, it is important
to first +no) the mechanism that causes sleep% .'enosine, a $anthine, accrues in the
acti*e (rain an' is only inhi(ite' (y a'enosine receptors locate' on the cells of the (asal
fore(rain that function in arousal% Thus, 'ro)siness, an' su(sequently, sleep, occurs as a
result of the slo)ing 'o)n of ner*e acti*ity (y the (in'ing of a'enosine#to#a'enosine
receptors in the fore(rain%
/
Caffeine, li+e a'enosine, (elongs to the chemical group
$anthine an' is so similar in structure to a'enosine that it can fit into a'enosine receptors%
Caffeine, ho)e*er, cannot simulate a'enosine an', as a result, can only ser*e to act as an
3
Sarnat, ;%B%, ? @ets+y, M% G% 4Aec 20025 Ae*elopment an' Aysgenesis 94/5=2/0#20!
4
Brain, M, ? Bryant, C% &% 4 .pr 20005% How Caffeine Works. ;o)Stuff&or+s%com%
Bhttp=66health%ho)stuff)or+s%com6caffeine%htmC Retrie*e' on 20 March 200%
antagonist against a'enosine%
0
Therefore, instea' of slo)ing 'o)n the ner*e acti*ity, the
(loc+ing of a'enosine (y caffeine 'oes Dust the opposite an' increases ner*e acti*ity
gi*ing the characteristic (oost of energy often associate' )ith caffeine consumption% 1n
general, (oth chemistry an' (iology ta+e an equal interest in the effects of chemical
su(stances on the form an' physiology though they may 'o so for 'ifferent reasons% 1t is
thus important that they are a(le to carry out e$perimental la(oratory research in or'er to
in*estigate, analy:e an' test hypotheses in an attempt to pro*i'e ans)ers to )hy an' ho)
things occur in the natural )orl' an' offer possi(le solutions to any pro(lems pose'%
Because the human species share at least some 'egree of conser*ation (et)een other
species through the genomes, it is often (eneficial to human (eings to use lo)er species,
as mo'els to (etter un'erstan' our o)n species%
This e$periment analy:es an' see+s to manipulate the (eha*ioral acti*ity of
planarians through operant con'itioning% The theory of operant con'itioning intro'uce'
(y psychologist, B% -% S+inner, is (ase' on the i'ea that Elearning is a function of change
in o*ert (eha*ior%F
8
Gperant con'itioning relies on en*ironmental interactions an' the
a''ition of a consequence to shape *oluntary (eha*ior% The consequence, usually in the
form a stimulus ser*es as positi*e reinforcement in shaping (eha*ior% 1n this e$periment
the planarians are e$pecte' to ElearnF as a result of recei*ing a stimulus of a shoc+ e*ery
time they (eha*e contrary to )hat they are (eing Etaught%F
Materials
5
@ehlig, .%, Aa*al, H%I%, ? Ae(ry, G% 4JJ25 Caffeine an' the central ner*ous system=
mechanisms of action, (iochemical, meta(olic an' psychostimulant effects% Brain Res
Brain Res Re*% 17425=!J#"0
6
Kearsley, G% 42005% Operant Conditioning (B.F. kinner!% The Theory 1nto Practice
Aata(ase% Retrie*e' from http=66tip%psychology%org
Planarian K#tray 4train#a#tray5 ma:e, (ro)n planarians 4Dugesia tigrina5, eggs, pipettes,
paint(rush, spring )ater, 8 *olt (attery, alligator clips, hol'ing containers, 0%0!M stoc+
solution of caffeine
E'perimental (esign
Planarians li*ing in fresh spring )ater )ere +ept in a sha'e' area at room temperature%
Planarians )ere teste' in the K#ma:e of a Train#.#Tray, i'entical to the tray illustrate' in
Fig. " (elo)% The tray, li+e its name suggests is in the shape of a EK,F )ith one electro'e
for each path)ay 4arm of the EKF5, an' t)o a''itional electro'es in the center of the (ase
arm EK%F 1n a''ition to the K#ma:e, the Train#.#Tray contains hol'ing areas for the
planarians, as )ell as positi*e an' negati*e attachment regions to )hich the 8 L (attery
attaches% The planarians )ere transferre' to an' from the Train#.#Tray )ith the use of a
fine#tippe' tooth(rush%
Fig. " illustrates a Train#.#Tray )ith the K ma:e, electro'es, an' hol'ing cells%
"
The ma:e )as line' )ith some of the spring )ater in )hich the planarians li*e' in or'er
to facilitate the mo*ement of the planarians% The planarians )ere fe' har'#(oile' egg
yol+ t)o 'ays prior to testing for each trial an' )ere fe' in )ee+ly inter*als thereafter%
7
Auane, M%, -orten(erry, G% ? ;oro)it:, .% 4JJ75 1nquiry in science using an animal
(eha*ior mo'el% Bhttp=66)))%)oo'ro)%org6teachers6(i6JJ76planaria6stu'entla(%htmC
Method
1n the first part of the e$periment, a group of 20 planarians )ere teste'% The pre#
con'itional perio' of the e$periment consiste' of running each planarian in the sample
through the ma:e ten times consecuti*ely, one after the other to 'etermine )here each
planarian ha' a natural ten'ency to selecti*ely choose one path o*er another% . pre#
con'itional perio' refers to a perio' in )hich the testing stimulus is not yet intro'uce'% 1n
this case, since the testing stimulus is a shoc+, no shoc+s )ere a'ministere'% Airectional
(ias, the ten'ency for the planarian to unconsciously choose one path o*er another, )as
'etermine' 'uring the #'ay pre#con'itional perio'% The testing for 'irectional (ias
in*ol*e' running each planarian through the ma:e ten times unai'e' (y any factor to
compose one trial% >ach time the planarian tra*ele' the ma:e an' )ent either left or right
)as consi'ere' a run% The 'irectional (ias )as 'etermine' only after each planarian
tra*ele' in the same 'irection, either left or right, for the maDority of the runs 48605%
Planarians that, for )hate*er reason, 'i' not sho) 'irectional (ias, that is, 'i' not tra*el
in the same 'irection for the maDority of the trials )ere 'iscar'e'% Gnce the planarian
complete' the testing, those that )ere not 'iscar'e' )ere la(ele' the control group an'
)ere separate' (y 'irectional (ias, so that three hol'ing containers )ere create'= one for
planarians )ith a 'irectional (ias to the right, one for planarians )ith a 'irectional (ias to
the left an' one for planarians )ith no 'irectional (ias% The planarians )ere then
containe' in their respecti*e containers (ase' on 'irectional (ias for the remain'er of the
e$periment% .fter 'irectional (ias )as 'etermine' for each planarian in the pre#
con'itional perio', the planarians )ere su(Decte' to a 0#'ay con'itioning perio' to
in*estigate learning, )hich in*ol*e' the planarians (eing traine' to tra*el in a 'irection
opposition to their 'irectional (ias% ;ence, a planarian )hose 'irectional (ias )as right
)oul' (e traine' to go left% The training in*ol*e' the each planarian recei*ing a stimulus
of a shoc+ )hen it 'e*iate' from the going in the 'irection opposing the 'irectional (ias%
The shoc+ )as a'ministere' through connections in the K#tray connecte' to a 8 *olt
(attery% -or each of the fi*e consecuti*e 'ays of the con'itioning 4or learning5 perio' the
planarian )oul' (e run through the ma:e in one trial, )hich consiste' of 0 runs, an'
shoc+ )oul' only (e applie' )hen necessary, that is, planarians go any)here (ut in the
'irection they )ere (eing traine' to go% The planarians )ere consi'ere' traine' )hen
they )ent in the 'esire' 'irection for a maDority of the ten runs 48605 for the maDority of
the 0#'ay training perio' 4!605% The application of shoc+ )as o(ser*e' from the
planarianMs response of contraction an'6or cessation of mo*ement%
.fter the 0#'ay con'itional perio', the planarians that sur*i*e' the training )ere
su(Decte' to a 0#'ay post#con'itional perio' of testing to in*estigate memory retention%
The (asis for 'etermining retention in the planarians in*ol*e' running each planarian
through the ma:e unai'e' (y any stimulus an' recor'ing ho) many times the planarian
tra*ele' in the correct 'irection o*er the 0 'ays% The planarians )ere consi'ere' to ha*e
EforgottenF )hat they ha' Elearne'F on the 'ay )hen they )ent in the 'irection of their
'irectional (ias for the maDority of the 0 runs%
The entire process )as repeate' )ith the only 'ifference (eing that for each trial,
0 planarians )ere no) e$pose' to 'iffering concentrations of caffeine concentration in
the 0#'ay con'itional perio'% The stoc+ solution of %0!$0
#
M caffeine )as first use'%
The planarians of each e$perimental group )ere to (e e$pose' to caffeine for ten minutes
after the pre#con'itional perio', (ut (efore the con'itional perio'% The caffeine )as
'ilute' )ith spring )ater to %0!$0
#2
M% The starting concentration for the caffeinate'
e$periments (ecame %0!$0
#/
M caffeine after a series of 'ilutions an' e$perimentation,
(ecause unli+e the other solutions, it 'i' not +ill the planarians% The solution )as 'ilute'
to pro'uce concentrations of 0$0
#0
M caffeine, %0!$0
#0
M caffeine an' 0$0
#8
M
caffeine, respecti*ely% .t the 'iffering le*els of concentration the proce'ures to test for
learning an' retention )ere carrie' out )ith the results (eing recor'e'% . total of "!
planarians )ere teste' o*er a "#)ee+ perio'%
)esults
Planarian group Time it too+ to
ElearnF on a*erage
4'ays5
Time it too+ to
EforgetF on a*erage
4'ays5
Sur*i*al Rate of
Planarians
Control !%8 2%J 060
%0!$0
#/
M caffeine !%/ 2%7 /60
0$0
#0
M caffeine /%2 2%7 060
%0!$0
#0
M caffeine !%7 !%7 860
0$0
#8
M caffeine !% /% 760
The control group too+ an a*erage of !%8 'ays to 'emonstrate learning% The
planarians e$pose' to %0!$0
#/
M caffeine too+ an a*erage of !%/ 'ays to learn, )hile
planarians teste' in 0$0
#0
M caffeine an' %0!$0
#0
M caffeine too+ an a*erage of /%2
'ays an' !%7 'ays, respecti*ely, to 'emonstrate learning% The planarians teste' in 0$0
#8
M
caffeine too+, on a*erage, !% 'ays to 'emonstrate learning%
1n regar's to retention, the control group of planarians retaine' )hat they ha'
learne' for a perio' of 2%J 'ays% The retention perio' for planarians teste' in %0!$0
#/
M
caffeine an' 0$0
#0
M caffeine )as 'etermine' to (e !%7 'ays for (oth groups% 1n %0!$0
#
0
M caffeine, the planarians teste' sho)e' the a(ility to retain E+no)le'geF for !%7 'ays
an' for the planarians teste' in 0$0
#8
M caffeine, /% 'ays%
The (eha*iors of the planarians )ere also o(ser*e' an' recor'e' throughout the
trials in or'er to trac+ any irregularities among the )orms% <nresponsi*eness )as also
recor'e' for further in*estigation% @ormal mo*ement of the planarians )as o(ser*e' as
stea'y elongation to propel themsel*es for)ar'% ;o)e*er, upon recei*ing the shoc+s,
planarians contracte' their (o'ies an' )ere slo) to (egin mo*ing again% .fter running
the planarians through the ma:e a num(er of times, some of the planarians (ecame
lethargic an' slo) mo*ing% Gthers e$hi(ite' scre)#li+e motions after (eing remo*e'
from their original en*ironments, an', interestingly enough, 'ie' shortly after 'isplaying
such motions% Mo*ements such as spiraling )ere unique to certain planarians an' )ere
often an in'icator a rapi'ly approaching 'eath% .fter (eing intro'uce' to relati*ely high
caffeine concentrations, the scre)#li+e motion (ecame more common, )hich implies that
caffeine concentration 'oes in'ee' the ner*ous system% The most common 'ispositions of
the planarians are illustrate' (elo)%

4a%5 4(%5
4e%5 4f%5
Fig. "= Motor (eha*ior o(ser*e' in planaria= 4a5 normal (eha*ior9 4(5 scre)#li+e
mo*ements9 4e%5 hea' lift9% 4f%5 curling into (all 4photos ta+en (y Katrina Cusac+5%
>ffect of Caffeine Consumption on the Rate of Iearning an' Memory in Planarians
>ffect of Caffeine Consumption on the Rate of Sur*i*al in Planarians
(iscussion
Throughout this la(, the researcher 'etermine' that ma+ing your o)n la( is
e$tremely 'ifficult, fin'ing out ho) to measure these miniscule )orms an' ta+e precise
measurements is more 'ifficult than the researcher coul' ha*e e*er imagine'% Besi'es the
fact that this la( )as *ery rough an' not *ery organi:e', the stu'ent thought the la( )as
slightly a(o*e a*erage quality for )hat limite' materials the stu'ent ha' to )or+ )ith%
Pre*ious research on the effect of caffeine on learning an' memory confirms that caffeine
'oes affect learning an' memory (ecause it acts as a stimulant in the central ner*ous
system% The mechanism of caffeine in the (rain is con'uci*e to learning an' in some
)ays, memory% The inhi(ition of a'enosine, cause' (y caffeine consumption, results in an
increase in the release of the neurotransmitters, 'opamine an' glutamate% Aopamine
functions in learning, cognition, 'ecision#ma+ing *oluntary mo*ement, arousal an',
punishment an' re)ar'%
7
1t is the function of 'ecision#ma+ing that enforces the i'ea of
8
Best B% 42005 Brain #eurotrans$itters%
Bhttp=66)))%(en(est%com6science6anatmin'6anatm'0%htmlN'opamineC
operant con'itioning to change (eha*ior% Aopamine plays a role in controlling the flo) of
information in the (rain% Research illustrates that higher le*els of 'opamine in the (rain
correlate )ith persons ma+ing 'ecisions )ith more positi*e outcomes%
J

0
The results of
the research sho)e' that people )ho suffere' from Par+insonMs 'isease )ho )ere not
ta+ing their me'ication )ere more li+ely to ma+e 'ecisions )ith negati*e outcomes than
those )ho too+ their me'ication%>rror= Reference source not foun' 1nterestingly enough,
the me'ication for Par+insonMs 'isease increases the concentration of 'opamine in the
(rain% .long )ith its other functions, it is safe to say that 'opamine, an' hence caffeine
consumption, 'oes enhance learning%
1n regar's to memory retention, the neurotransmitters, acetylcholine an'
glutamate, play significant roles% 1ncrease' caffeine concentrations in the central ner*ous
system in'uce the release of acetylcholine in the hippocampus, the area of the (rain
associate' )ith memory control, i%e%, formation, storage an' retrie*al of memory%

.cetylcholine functions in the plasticity an' e$cita(ility of neurons, specifically in faster
reaction times an' short#term memory%
2
Ii+e, acetylcholine, glutamate also functions in
synaptic plasticity, the a(ility of the synapse (et)een neurons to change in strength% The
glutamate receptor, ##methyl D#aspartate 4@MA.5 receptor, functions in controlling
9
Goo'e, >% 4200/5% C% &esearc'ers find t'at dopa$ine is ke( to )earning )ikes and
dis)ikes% <ni*ersity of Colora'o at Boul'er%B http=66(iopsychiatry%com6'opamine6li+es#
'isli+es%htmlC
1

11
McIntyre$ C*$ Shanthi$ P* Marriott L* and +old

P* 4-e( 20025% Competition (et)een
Memory Systems= Acetylcholine Release in the ;ippocampus Correlates @egati*ely
)ith Goo' Performance on an .myg'ala#Aepen'ent Tas+ *'e +ourna) of #euroscience,
224!5="#"8
12
Milner, B%, Squire, I%R%, an' Kan'el, >%R%4JJ75 Cogniti*e neuroscience an' the stu'y
of memory% #euron 20=//0#/87
synaptic plasticity an' memory%
!
&hen (oun' to glutamate, the @MA. receptor
comple$ allo)s for the facilitate' release of Ca
2O
ions across the synapse%
/
The Ca
2O
ions
then (in' )ith calmo'ulin an' acti*ate calcium#calmo'ulin#'epen'ent +inase 11
4CaMK115% This mechanism, stimulate' (y increase' le*els of glutamate, ultimately lea's
tolong#term potentiation, a form of synaptic plasticity that causes long#lasting
enhancement in signal transmission (et)een t)o neurons, an' hence, enhances
memory%
0
The (in'ing of Ca
2O
an' calmo'ulin is regulate' (y autoinhi(ition an' so at
some point memory is controlle'%
8
;o)e*er, (ecause the half#life of caffeine *aries
)i'ely among in'i*i'uals, the effects of the 'rug on ITP also *aries% Aifferent stu'ies
sho) 'ifferent effects of caffeine on memory retention, ho)e*er, they all suggest that the
relationship (et)een caffeine an' memory retention is 'ose#'epen'ent )ith higher
concentrations impair long#term memory, )hile lo)er 'oses ha*e little to no effect on
memory retention%
"
-rom the results of this e$periment, it can (e 'etermine' that the group of
planarians that )ere e$pose' to the lo)est concentration )ere most successful at learning
an' retention as that group too+ the least amount of time on a*erage to ElearnF an' too+
the greatest time to EforgetF on a*erage% The results also sho) that at higher
13
-ei, I% ? Tsien, H%P% 4Huly 8, 200J5% Clinical 1mplications of Basic Research= Memory
an' @MA. receptors% #ew ,ng)and +ourna) of -edicine !8=!02,
14
.earning, -e$or( and .ong/ter$ 0otentiation. 4Han 2005%
Bhttp=66users%rcn%com6D+im(all%ma%ultranet6BiologyPages6I6ITP%htmlC
15
Coo+e, S%-% ? Bliss, T%L%P% 420085% Plasticity in the human central ner*ous system%
Brain #%, 4Pt "5= 80JQ"!%
Bhttp=66(rain%o$for'Dournals%org6cgi6content6a(stract62J6"680JC
16
Griffith, I% 4Sep 200/5% )egulation of Calcium-Calmodulin.(ependent Protein
/inase II Activation 0y Intramolecular and Intermolecular Interactions *'e +ourna)
of #euroscience, 2/4!J5=7!J/#7!J7 Bhttp=66neuro%cD(%net6cgi6content6full62/6!J67!J/C
17
&ar(urton, Aa*i' M 4JJ05% >ffects of caffeine on cognition an' moo' )ithout
caffeine a(stinence% 0s(c'op'ar$aco)og( ##,= 88Q"0%
concentrations yiel' (etter results up until a certain point at )hich, the lo)er
concentrations yiel' (etter results% 1t can also (e inferre' that, )ith the e$ception of the
control group lo)er concentrations of caffeine correlate )ith higher sur*i*al rates% The
fin'ings thus sho) that caffeine can ha*e a positi*e effect on learning an' memory
'epen'ing on the concentration of the caffeine% 1n fact, results sho) that there )as an
optimum concentration at )hich (oth learning an' memory retention reache' their pea+s%
Cases, in )hich the planarians 'ie' upon e$posure to caffeine, such as )ith the stoc+
solution of %0!$0# M caffeine an' its ten#fol' 'ilution, re*eal that high concentrations
of the caffeine solution can +ill a planarian%
1f the researcher coul' 'o this la( once more, the stu'ent )oul' change the en*ironment
to that of a real scientific la(, )here resources an' materials are rea'ily a*aila(le, such as
more planarian )orms, )hich )oul' increase the accuracy of the results% The stu'ent
)oul' also ma+e it so there is enough time to 'o the la(, not o'' times 'uring the school
'ay% Keeping the )ater at a perfect temperature an' controlling (acterial gro)th to the
en*ironment an' surroun'ings that the planarians are use' to resi'ing in )oul' ha*e
really helpe' the e$periment% 1nstea' of Dust testing the rate at, )hich the planarians
regenerate' in a caffeine 6 )ater solution, )hich the stu'ents e$periment trie' to 'o, there
)as also the time in )hich the planarians )oul' ha*e ha' to a'Dust to the )ater
temperature an' ne) forms of (acteria that the researcher coul' not factor in9 this, in
fact, playe' a maD or role in the e$peri ment that the stu'ent 'i', an' it
re'uce' the chance that the planarians ha' at sur*i*ing%