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The effects of an RNA polymerase in the improvement of verbal learning and memory
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The effects of an RNA polymerase in the improvement of verbal learning and memory
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THE EFFECTS OF AN RNA POLYMERASE IN THE IMPROVEMENT
OF VERBAL LEARNING AND M EM ORY
by
Augusto B r itto n
A D i s s e r t a t io n P resen ted to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In P a r t i a l F u lfillm e n t of th e
Requirements f o r the Degree
DOCTOR OF PHILOSOPHY
(Education)
February 1973
INFORMATION TO USERS
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Xerox University Microfilms
300 N orth Z a a b R oad
Ann Arbor, M ichigan 48106
73-18,798
BRITTON, A ugusto, 1932-
T H E EFFECTS O F A N R N A PO L Y M ER A SE IN T H E
IM PR O V E M E N T O F VERBAL LEARNING A N D M E M O R Y .
U n iv e r sity o f Southern C a lifo r n ia , Ph.D., 1973
P sy ch o lo g y , experim en tal
University Microfilms, A X ER O X Company, Ann Arbor, Michigan
C o p y rig h t © by
AUGUSTO BRITTON
1973
THIS DISSERTATION HAS BEEN MICROFLIMED EXACTLY AS RECEIVED
UNIVERSITY O F SOUTHERN CALIFORNIA
THE GRADUATE SCHOOL
UNIVERSITY PARK
LOS ANGELES. CALIFORNIA 8 0 0 0 7
This dissertation, written by
............................ AyQUSTO..BMTTON..........................
under the direction of h.Jk§.. Dissertation Com Â
mittee, and approved by all its members, has
been presented to and accepted by The Graduate
School, in partial fulfillment of requirements of
the degree of
D O C T O R OF P H IL O S O P H Y
Dram
Date F e b r u a r y 1 2 2 . 2
DISSERTATION COMMITTEE
^ < i
, Chairman
ACKNOWLEDGMENTS
During th e co n ception and conduction o f t h i s r e Â
search , many people encouraged, counseled and advised th e
au th o r. The a u th o r acknowledges, w ith l a s t i n g g r a t i t u d e ,
the h e lp fu ln e s s and g e n e ro s ity o f th e se people:
Dr. Newton S. M e tfe sse l, my committee c h a i r Â
man, and the o th e r members of my committee,
Dr. Prank H. Fox and Dr. Norman T ib e r, fo r
t h e i r support and s e r v ic e s provided.
The I n t e r n a t i o n a l Chemical and N ucleic
C o rp o ratio n of I r v i n e , C a l i f o r n i a , fo r p r o Â
v id in g th e drug Ribaminol and a ls o fo r t h e i r
t e c h n i c a l su p p o rt.
The a d m in is tr a tio n of th e Sepulveda VA
H o sp ita l fo r making a v a i l a b l e t h e i r s t a f f
and p a t i e n t s .
l i
CONTENTS
Page
LIST OF TABLES........................................................................................ iv
LIST OF F IG U R E S ................................................................................... V
Chapter
I . AN ANALYSIS OF THE PROBLEM...................................... 1
I n tro d u c tio n
The Evidence Im p lic a tin g RNA
in Learning and Memory
T h e o re tic a l Models E xplaining th e Role
of RNA In Learning and Memory
Form ulation of t h e Research Problem
Evidence S t i l l In co n clu siv e
D e fin itio n s of Some Terms
I I . REVIEW OF THE RELATED LITERATURE......................... 22
S tim u la tio n Tasks and P r o te in S ynthesis
P ro te in and N ucleic I n h i b i t o r s '
T ra n s fe r S tu d ies
RNA as an Inducer of Learning
Conclusions and R atio n ale
I I I . DESIGN AND METHOD........................................................... 47
V aria b le s
S u b jects
The Design
M a te ria l
Procedure
IV. R E SU L T S ................................................................................... 51
Data Analyses
D iscussion of R esu lts
Summary
V. SUMMARY, CONCLUSIONS, AND RECOMMENDATIONS . 58
Summary
Conelusions
Recommendations
REFERENCES.................................................................................................. 67
APPENDIX...................................................................................................... 76
i l l
LIST OP TABLES
Table Page
1. A nalysis of V ariance f o r th e Learning
C r i t e r i o n .................................. 52
2. A nalysis of V arian ce: R ecall........................................... 53
3. A nalysis of V arian ce: R ecognition .......................... 54
4. Mean Comparison f o r D ig it Span
S e le c tio n T e s t .................................................................... 7 6
5. Mean Comparison of Groups
b e fo re C rossover ................................................................ 76
6 . Mean Comparison o f Groups
a f t e r C r o s s o v e r ................................................................ 76
Iv
LIST OF FIGURES
Figure Page
1. A Genetic Model o f M emory............................................. 7
2. The Rappoport-Daginawala Model of
Memory S torage ................................................................ 9
3. C o n so lid a tio n and Maintenance of
Permanent Memory ........................................................... 10
v
CHAPTER I
AN ANALYSIS OF THE PROBLEM
I n tr o d u c tio n
This re se a rc h Is concerned w ith th e presumed r o le
of rib o n u c le ic a c id s (RNA) in th e improvement of le a rn in g
and memory.
Among th e p e rp le x in g problems th a t co n fro n t p sy Â
c h o lo g is ts to d ay is th a t of e x p la in in g th e mechanisms
whereby sensory ex p erien ces o f e x te r n a l and i n t e r n a l worlds
of th e in d iv id u a l are transform ed by th e organism in to
r e p r e s e n t a t i o n a l pro cesses t h a t symbolize e x p e r i e n t i a l
events during le a rn in g .
B io lo g ic a l th e o r ie s and e x p la n a tio n s o f th e pheÂ
nomena of le a rn in g and memory have c e n te re d around neuroÂ
lo g i c a l or biochem ical fo rm u la tio n s.
I t was Katz and H alste ad (1950), who o r i g i n a l l y
suggested t h a t m acromolecular s tr u c t u r e s might be resp o n Â
s i b l e f o r c a rry in g in fo rm a tio n and s to r in g memory. These
r e s e a rc h e rs claim ed t h a t m olecules were formed and t h a t
th e s tim u la tio n of n e u ra l t i s s u e by im pulses during l e a r n Â
ing events caused th e s e m olecules to assume a s p e c if ic
c o n f ig u ra tio n c o r r e l a t e d w ith a given ex p erien ce.
1
2
These e a r ly fo rm u latio n s g en erated a f r e n e t i c
search among re s e a r c h e r s in d i f f e r e n t p a r t s of th e world
f o r th e "code" of p sy c h o lo g ic a l memory. As a consequence,
v ast amounts of e m p iric a l evidence has been g a th e re d , some
su p p o rtin g and some r e f u tin g th e se t h e o r e t i c a l fo rm u latio n s.
The Evidence Im p lic a tin g RNA
in Learning and Memory
During th e l a s t 20 y e a rs, t h e r e has been a v a s t
amount of re se a rc h done to determ ine th e r o le of RNA in
le a rn in g and memory. The p re se n t re s e a rc h i s concerned
w ith th e evidence from four sources:
1. From s e p a ra te i n v e s t i g a t o r s using d i f f e r e n t types
of s tim u la tio n ta s k s in which changes in RNA conÂ
te n t and base r a t i o s in anim als were found;
2. From s tu d ie s In v o lv in g p r o te in s y n th e s is i n Â
h i b i t o r s and b e h a v io ra l i n d i c a t o r s o f such
i n h i b i t o r y e f f e c t s ;
3. From t r a n s f e r s tu d ie s in v o lv in g th e i n j e c t i o n
of RNA e x t r a c t s co n ten t from th e b ra in of
" tra in e d " r a t s to th e b ra in of "naive" r a t s ;
and
4. From s tu d ie s in v o lv in g the in g e s tio n o f subÂ
sta n c e s which in c re a s e th e p ro d u c tio n of
neuronal RNA and are evidenced by b e h a v io ra l
measures in d i c a t i n g le a rn in g f a c i l i t a t i o n .
3
T h e o re tic a l Models E xplaining th e Role
of RNA In Learning and Memory
The RNA Model
The development of th e th e o ry t h a t th e RNA molecule
I s th e organic s u b s t r a te of memory, o r t h a t a t l e a s t I t
p lay s a unique r o le In th e encoding p ro c e ss, I s c h i e f l y
connected w ith Hyden's (1959» 1961, 1962) works. During
th e l a s t 20 y e a rs, Holgar Hyd£n o f Sweden has been conductÂ
ing e x te n siv e re se a rc h ex p lo rin g th e r o le of RNA and p ro Â
t e i n s In le a rn in g and memory. In th e s e f i r s t p u b lic a tio n s ,
he showed th a t th e a d e n in e /u r a c il r a t i o of th e RNA In th e
c e l l nucleus of r a t n e u ra l t i s s u e In c re a s e d s i g n i f i c a n t l y
follow ing a le a rn in g experim ent. I t was a lso s t a t e d t h a t
an In c re a se d amount of RNA p e r neuron could be observed In
r a t s which had e s ta b lis h e d a le a rn e d p a t t e r n o f sensory
and motor a b i l i t i e s . In e a r ly t h e o r i e s , Hyden proposed
th e h y p o th esis which s t a t e d t h a t a t th e fou n d atio n o f the
memory p ro c e ss, th e r e I s a change In t h e bases In th e RNA
m olecule. This a l t e r a t i o n o f base sequence and com position
i s brought about by th e a c tio n of th e frequency modulated
e l e c t r i c a l s ig n a ls re c e iv e d by th e neuron and I t s d e n d r ite s .
The new p a t t e r n in th e RNA m olecule th e n remains and s p e c iÂ
f i e s th e p roduction of a new p r o te in . Now subsequent
e l e c t r i c a l s tim u la tio n produces ra p id d i s s o c i a t i o n o f the
newly s p e c if ie d p r o te in . Follow ing t h i s a c tio n , th e d is s o -
4
e la te d p r o te in combines w ith a complementary m olecule formÂ
in g a complex o r h y b rid s t r u c t u r e which In flu e n c e s th e r e Â
le a s e of t r a n s m i t t e r su bstance In th e synapse so th a t the
p o s ts y n a p tlc neuron I s e x c ite d . In summary, o f Hyden's
h y p o th e s is , th e permanent change In th e RNA molecule Is a
response to a c h a r a c t e r i s t i c e l e c t r i c a l Input (frequency
modulated waves) d e s c rib in g a sensory e x p erien ce; when a
neuron In an a lre a d y e s ta b lis h e d memory t r a c e I s a c tiv a te d ,
th e im pulses s tim u la te th e p r o te in ( s p e c if ie d by th e memory
RNA) and r e l e a s e th e t r a n s m i t t e r su b stan ce causing th e
next neuron in th e c h a in to be e x c ite d .
The problem w ith t h i s h y p o th e sis i s t h a t Hyden did
not show how an a f f e r e n t im pulse can a l t e r tem plate p ro Â
t e i n s of DNA so as to perm it s y n th e s is o f unique RNA fo r
each b i t o f in fo rm a tio n tra n s m itte d by th e impulse (RappoÂ
p o rt & Daginawala, 1970).
More r e c e n t l y , Hyden (1964, 1970) has advocated
th e involvement o f g l i a l c e l l s . While th e e a r l i e r model
d id not in v o lv e th e g e n e tic ap p aratu s in th e encoding p ro cÂ
e s s , Hyden's l a t e r th e o ry proposed t h a t RNA from g l i a e n te r
th e neuron and a c t as d e re p re s s io n ag en ts to f r e e a gene o r
genes f o r th e p ro d u c tio n of a s p e c i f i c p r o te in which th en
in flu e n c e s sy n a p tic c o n d itio n .
The Genetic Model
Concerned w ith th e need to p re se rv e th e concept of
a u n ity o f biochem ical d ev ices o f c e l l u l a r c o n tr o l from th e
h ig h e s t to th e low est c e l l s , Barondes (1964) and Bonner
( 1 9 6 6), among o th e r s , proposed t h a t the n e c e ssa ry mechanism
f o r memory must f i t i n to th e f u n c tio n a l scheme of th e g e Â
n e t i c a p p a ra tu s.
Bonner ( 1 9 6 6), p o s tu la te d a model fo r memory which
in v o lv e s th e f e a t u r e s of th e "sw itching" mechanisms which
have been found to be a t th e h e a r t of th e o p e ra tio n of th e
re a d in g out o f th e g e n e tic code. In t h i s th e o ry , th e
chemical a l t e r a t i o n of the neurons r e s u l t i n g in a memory
i n s c r i p t i o n depends upon th e d e re p re s sio n of a p r e v io u s ly
re p re sse d gene o r genes. These genes, now fre e to fu n cÂ
t i o n , m anufacture su b stan ce p ro v id in g f o r th e passage of
e l e c t r i c a l im pulses through s p e c i f i c synapses. In t h i s
p o in t of view, as w e ll as t h a t adopted by Barondes (1964),
t h e r e are no memory m olecules as such, but In s te a d memories
a re s to re d as f a c i l i t a t e d s y n a p tic c o n d itio n s . These new
sy n a p tic co n n e c tio n s come about e i t h e r by v i r t u e of th e
p ro d u c tio n o f a s p e c i a l p r o te in o r by th e s t r u c t u r a l modiÂ
f i c a t i o n of a lre a d y e x i s t i n g p r o te in s . B onner's model,
which su g g ests t h a t any RNA sy n th e siz e d follo w in g le a rn in g
should be a gene p ro d u c t, p r e d i c t s th a t th e le a r n in g -
induced RNA can be hy b rid ed w ith DNA. By c o n t r a s t , RNA
6
which has not been a l t e r e d by th e g e n e tic DNA could not be
hy b rid ed w ith DNA. The reason f o r t h i s i s t h a t th e RNA
forms as a h y b rid complex m olecule w ith one s tra n d of th e
d iv id e d , form erly two stra n d e d , DNA m olecule. In summary:
Memory i s a s p e c ia l ad ap tio n in th e neuron o f g e n e ra l re g u Â
l a t o r y mechanism o f th e g e n e tic "sw itching" system . The
i n s t r u c t i o n s f o r th e s y n th e s is o f a m em ory-specific p r o te in
a re co n ta in e d in a passage of th e g e n e tic codebook, l i k e a
d i g i t a l computer program. This passage can be read out by
the d e re p re s s io n of a r e p r e s s o r m olecule. The r e p r e s s o r
molecule which keeps th e program form e x p re ss io n , a c ts by
combining r e v e r s i b l y w ith an o p e ra to r gene. As i n d ic a te d
in F ig u re 1, th e d e re p re s sin g m olecule i s seen as somehow
being a c t i v a t e d by th e impinging e l e c t r i c a l im pulse. The
operon i s a s e t o f genes sw itched on by th e o p e ra to r to
sy n th e s iz e RNA which, i n tu rn , d i r e c t s th e s y n th e s is o f
c e l l u l a r p r o te in . Note t h a t th e mechanism d i r e c t l y conÂ
n e c tin g th e im pinging e l e c t r i c a l im pulses w ith th e molecuÂ
l a r a l t e r a t i o n s remains unexplained in t h i s model. Also
th e co n n ectio n between RNA or " s p e c if ic " p r o t e i n and th e
e l e c t r i c a l im pulse response of th e neuron to s tim u la tio n
is u n ex p lain ed .
REGULATOR
D N A
OPERATOR OPERON
OOCpeOQQBOOO
REPRESSOR
M O L E C U L E S
AvWW
J L QJ 2 J ZJ Z rn a
DEREPRESSOR
M O L E C U L E S
M E M O R Y
SP E C IF IC P R O T E IN
.b ’ig . 1. A g e n e tic model of memory
8
Other R elated T heories
L andauer's e x p la n a tio n s . S im ila r t o th e model
advocated by Hyden i s t h a t of Landauer (1964), which specÂ
u l a t e s th a t th e b a sic event of c o n d itio n in g o r le a rn in g i s
th e t r a n s f e r of RNA m olecules from surrounding g l i a l c e l l s
i n t o conducting neurons and subsequent p a r t i c i p a t i o n of
t h i s RNA as a messenger RNA from p r o t e i n s y n th e s is in
neurons. The t r a n s f e r of RNA a c ro ss membranes of a c t iv a te d
neurons during im pulse conductions i s presumed to be due
to th e removal o f a b a r r i e r c o n s is tin g of an u n fav o rab le
p o t e n t i a l g r a d ie n t which i s r e p re s e n te d by membrane imÂ
p e rm e a b ility .
The Rappoport-Daginawala m odel. I n th e quest to
f in d a model which can be e m p iric a lly v a lid a te d , Rappoport
and Daginawala (1970), i n j e c t e d Actinomycin D, a p r o te in
i n h i b i t o r , in t o th e b r a in o f c a t f i s h , and found a d e c re a se
i n n u c le a r RNA by 7-12 p e rc e n t when compared to f i s h b r a in
w ith Actinomycin D. However, th e p resence of A ctin o Â
mycin D did not i n t e r f e r e e i t h e r w ith th e s y n th e s is of or
th e b a s e - r a t i o change i n b r a in n u c le a r RNA s y n th e s is during
o l f a c t o r y s tim u la tio n by m orpholine. Based on t h e i r f i n d Â
in g s and t h a t of o th e r s , th e s e r e s e a r c h e r s proposed a model
f o r memory sto ra g e as fo llo w s:
From th e e x i s t i n g r e p o r ts in th e l i t e r a t u r e and from
th e r e s u l t s o b ta in e d i n th e p re s e n t s tu d i e s , th e conÂ
c e p tu a l p ro cess o f in fo rm a tio n a c c ru a l i n th e b ra in
9
i s v is u a l i z e d as fo llo w s: The coded a f f e r e n t im pulse
i s tr a n s m itte d along th e axons and d e n d r ite s o f th e
neurons, and th u s a f a i r number of neurons and g l i a
r e c e iv e th e same in fo rm a tio n . Sim ultaneously, t h i s
s ig n a l p a sse s through t h e cytoplasm to th e nu cleu s
w ith in th e b ra in c e l l s . As th e a f f e r e n t im pulse enÂ
t e r s th e n u cleu s, i t i s tra n sd u ced in some unknown
manner so t h a t RNA s y n th e s is occurs in such a manner
t h a t th e sequence of bases corresponds to th e coded
in fo rm a tio n i n th e im pulse. Thus, th e coded a f f e r e n t
im pulse induces th e s y n th e s is o f RNA whose base s e Â
quence c o n ta in s th e in fo rm a tio n tra n s m itte d by t h e
im pulse. From th e d a ta p re se n te d h e re , th e newly
s y n th e s iz e d RNA r e p re s e n ts s h o rt-te rm memory." I t
i s f u r t h e r c o n c e p tu a liz e d t h a t t h i s RNA su b se q u e n tly
a c t s as a m essenger RNA which d i r e c t s b io s y n th e s is o r
a p r o te in whose amino a c id sequence i s determ ined by
th e base sequence i n t h i s RNA. Thus, newly s y n th e s iz e d
p r o te in r e p r e s e n ts th e long-term in fo rm a tio n s to r a g e
u n it o r engram [p. 487].
AFFERENT
IMPULSES
AMINO
ACIDS
RNA SYNTHESIS
PROTEIN SYNTHESIS
EFFERENT
IM PULSES
SC NSC*
STORAGE UNIT
STORAGE COMPLEX
F ig . 2. The Rappoport-Daginawala model o f memory
sto ra g e (from Rappoport-Daginawala, 1970).
10
The H alstead-R ucker model. In o rd e r t o account f o r
th e r o le o f s h o rt-te rm and lo n g -term memory, H alste ad and
Rucker ( 1 9 6 8), based on t h e i r own r e s e a rc h , and t h a t of
o th e r s (among o th e r s : Davis & A granoff, 1966; F lex n er et
a l . , 1967) proposed th e fo llo w in g model:
Organism Neuron M olecular l e v e l
Stim ulus input ----------- + â– Impulse code â–º I n i t i a l inducing
agent
I
Response output < -
D N A
m R N A
Impulse code <â– P r o tein
F ig. 3. C o n so lid a tio n and m aintenance of permanent
memory. Two feedback loops may have evolved to p r o te c t
permanent memory a t th e m o lecu lar le v e l (H alstead & Rucker,
19 6 8).
As in d ic a te d i n F ig u re 3* th e b a sic c h a r a c t e r i s t i c of t h i s
model I s th e second tnacromolecular feedback loop which may
have evolved to m a in ta in and r e g u la te an adequate le v e l of
p r o te in s e s s e n t i a l f o r t r a c e r e t r i e v a l and f o r p r o te c t io n
of th e ch ain a t i t s weakest l i n k . This model has th e adÂ
vantage over o th e rs in t h a t th e lay o u t i s sim ple and i t
s p e c i f i e s th e messenger RNA as being involved In th e conÂ
s o l i d a t i o n phase o f memory. But s t i l l th e q u e stio n r e Â
mains, what i s t h a t s p e c i f i c macromolecule t h a t fu n c tio n s
11
as th e r e g u la to r molecule? H alste ad and Rucker have not
answered t h i s q u e s tio n , so we co n tin u e probing.
The r e g u la to r molecule t h a t c o n tr o ls th e ex p ressio n
of th e memory co d e. I f we accept th e assum ption th a t one
of th e n u c le ic a c id s I s a memory m olecule, th e q u e stio n I s ,
how Is I t re g u la te d ? I t Is suggested t h a t t h i s molecule
r e g u l a t o r may be a p r o te in . Huang and Bonner ( 1962) found
w ith pea embryo chrom atin t h a t when th e h is to n e f r a c t i o n of
DNA was removed, th e r a t e of RNA s y n th e s is in c re a se d f i v e Â
fo ld . These re s e a r c h e r s concluded t h a t th e fu n c tio n of
h is to n e was to bind DNA and block th e t r a n s f e r of inform aÂ
t i o n from DNA. G aito (1963)* r e f e r r i n g to th e is s u e of th e
locus o f memory, suggested th a t during s tim u la tio n , th e
c o n f ig u ra tio n of th e h is to n e s i s m odified as to make a v a i l Â
a b le th e p o t e n t i a l in h e re n t in th e n u c le ic a c id s . This
m o d ific a tio n would a l t e r th e n u c le ic a c id - h is to n e complex
and would re p re s e n t th e symbolic r e p r e s e n t a t i o n of th e exÂ
p e r i e n t i a l event. In summary, a f t e r th e review of th e se
d i f f e r e n t t h e o r e t i c a l models, th e r e s e a r c h e r has not found
d i r e c t evidence to s e l e c t one o f th e se models as th e model
t h a t i s ab le to account f o r th e locus of memory.
C r i t e r i a f o r a Model
W e need a s e t of c r i t e r i a on which to b u ild a t h e Â
o r e t i c a l model in o rd e r to account f o r a permanent memory
12
t r a c e . Dingman and Sporn (1967) > o ffe re d th e fo llow ing
c r i t e r i a f o r th e s e l e c t i o n of a giv en m olecule, a s e t of
m olecules, and a s tr u c t u r e o r s e t of s t r u c t u r e s as a p e r Â
manent memory t r a c e : ( l) I t must undergo a change o f s t a t e
In response to th e ex p erien ce t o be remembered; (2) th e
a l t e r e d s t a t e must p e r s i s t as long as th e memory can be
dem onstrated; and ( 3 ) s p e c i f i c d e s tr u c t i o n of th e a l t e r e d
s t a t e must r e s u l t In permanent lo s s of memory. U n til
th e se c r i t e r i a a re met, any experim ental d a ta r e l a t i n g RNA
and memory are h ig h ly c i r c u m s t a n t i a l .
Form ulation of th e Research Problem
Evidence S t i l l In c o n c lu siv e
So f a r no c o n c lu s iv e , d i r e c t evidence has been
found to i n d i c a t e t h a t RNA o r any o th e r molecule i s the
source of memory. As i t w i l l be in d ic a te d in C hapter I I ,
th e fin d in g s im p lic a tin g RNA s u f f e r from a v a r i e t y o f conÂ
founding f a c t o r s t h a t i n v a l i d a t e t h e i r c o n c lu sio n s. Thus,
th e q u e stio n , can RNA improve human v e rb a l le a rn in g and
memory, s t i l l rem ains unanswered.
The Hypotheses
There i s an o v e r a l l , g e n e ra l h y p o th e sis t h a t t h i s
re s e a rc h i s p robing: RNA i s not th e source of memory s t o r Â
age as suggested by many r e s e a r c h e r s . In o rd e r to a r r i v e
a t t h i s d ed u ctio n , th e s t r a t e g y Is t o fo rm ulate a hypotheÂ
13
s i s t h a t i s t e s t a b l e . In t h i s s p e c i f i c c ase, th e hypotheÂ
ses a re t h a t a s y n th e tic RNA polym erase, ta k e n o r a l l y ,
does not I n c re a s e :
Human v e rb a l le a rn in g sco re s on a p a lr e d -
a s s o c ia te s ta s k .
Ho 2
R e c a ll sco re s on a P-A ta s k .
H o3
R ecognition scores on a P-A ta s k .
Assumptions on which t h i s
H ypothesis I s Based
The r o l e o f RNA so f a r , as i n d ic a te d by experimenÂ
t a l r e s e a r c h , i s :
1. To r e g u la te th e p ro d u c tio n of s t r u c t u r a l p ro Â
t e i n s (N irenberg, 1 9 6 5).
2. To m ediate th e p ro d u c tio n and r e g u l a t io n of
enzymes (N irenberg, 1965; Rich, 1 9 6 5).
3. To f a c i l i t a t e s y n a p tic tra n s m is s io n (Appel
e t a l . , 1970; A granoff, 1970).
And as such, i t s in flu e n c e on th e CNS, th e neuron, axon,
d e n d r i te s , and synapse i s of a su p p o rtiv e , r e p le n is h in g ,
f a c i l i t a t i v e n a tu re and not t h a t of a memory m olecule.
Ho.
14
Q uestions to be Answered
I n t h i s ResearcK
1. Can a s y n th e tic RNA, Ribaminol, tak en o r a l l y
Improve v e rb a l le a r n in g and memory of th e aged?
2. Is t h i s r e s e a r c h paradigm e f f i c i e n t and powerÂ
f u l In c o n t r o ll i n g extraneous f a c t o r s th a t
could I n v a l i d a t e th e e f f e c t s of th e e x p e r iÂ
m ental tre a tm e n t, and does I t lend I t s e l f
f o r r e p l i c a t i o n ?
S ig n ific a n c e and Im p lic a tio n s
o f t h i s Research
I f th e n u l l h y p o th e sis of no e f f e c t of RNA In th e
Improvement of human v e rb a l le a rn in g I s not r e j e c t e d :
1. Then we w i l l have an answer i n d ic a tin g th a t
RNA, under th e s e s p e c if ic c o n d itio n s , does
not f a c i l i t a t e v e rb a l le a rn in g and memory.
2. This paradigm could be r e p l i c a t e d In a wide
range o f s tu d ie s r e l a t e d to v e rb a l le a r n in g .
On th e o th e r hand, I f th e n u l l h y p o th e sis I s r e Â
je c te d and I f In f a c t RNA I s capable of enhancing behavÂ
i o r a l components of human v e rb a l le a rn in g , under th e s e
s p e c i f i c c o n d itio n s ; i t should prove to be a v ery p o te n t
t o o l In th e s o l u t i o n of some o f th e s o c i a l and e d u c a tio n a l
problems t h a t vex th e concerned b e h a v io ra l s c i e n t i s t .
B r i e f ly , b e n e f i t s from p o s i t i v e fin d in g s could be:
15
1. The a r r e s t of le a rn in g and memory d e t e r i o r a t i o n
i n th e aged.
2. The improvement of th e g e n e ra l i n t e l l i g e n c e le v e l
o f a l l people.
3. The improvement of th o se in d iv id u a ls who f a l l on
th e lower end o f th e IQ s c a le [Krech, 1967].
4. The a c t u a l i z a t i o n of th e change in s p e c i f i c a b i l -
i t i e s which i s becoming a re a c h a b le r e a l i t y
Lkrech, 1967].
5 . The c o r r e c tio n of some o f th e c o n g e n ita l f a c t o r s
which im p air c o g n itiv e a b i l i t i e s by app ly in g p r e Â
n a t a l tre a tm e n t o f RNA which a c t s as a m ediator
i n th e p ro d u c tio n of p r o te in and o th e r biochem iÂ
c a l su b stan ces of v i t a l im portance f o r th e d eÂ
velo p in g organism [Scrimshaw & Gordon, 1968].
D e lim ita tio n s
The p re se n t r e s e a rc h was lim ite d to male g e r i a t r i c
p a t i e n t s between th e age range of 50-80 w ith some degree of
b r a in impairment as in d ic a te d by t h e i r m edical re c o rd s from
th e Sepulveda V eterans H o s p ita l. A s in g le dose (250 mg)
of th e drug Rlbaminol was given o r a l l y . The le a rn in g ta s k
was lim ite d to e ig h t p a i r s of P a ire d -A s so c ia te s words f o r
each tre a tm e n t c o n d itio n and Ss_ were t e s t e d f o r l e a r n i n g ,
r e c a l l , and r e c o g n i t io n .
D e f in itio n s o f Some Terms
Acetoxycyclohexlm lde (AXM). A drug t h a t i n h i b i t s
p r o te in s y n th e s is a t th e t r a n s l a t i o n a l le v e l by p re v e n tin g
th e t r a n s f e r of th e amino a c id from th e tRNA i n t o th e pepÂ
t i d e c h a in .
16
Actinomycin D. A drug t h a t blocks t r a n s c r i p t i o n
of DNA so t h a t RNA s y n th e s is c e a se s, but does not seem to
a f f e c t nerve a c t i v i t y .
Amnesia. A p a r t i a l o r t o t a l lo s s of memory.
Components of DNA. The fo u r bases a re : adenine,
guanine, thymine and c y s to s in e (sym bolized as A, Q, T, C).
Components of RNA. RNA i s s im i l a r to DNA except
t h a t RNA c o n ta in s rib o s e in s te a d of deoxyribose and th e
base u r a c i l in s te a d of thymine.
C o n s o lid a tio n . A p ro cess t h a t i s h y p o th esize d to
tak e p la c e a f t e r exposure to a le a rn in g t r i a l o r to an ex Â
p e rie n c e . C o n so lid a tio n i s presumed n e c e ssa ry i f the
e f f e c t of th e ex p erie n c e i s to become permanent.
C y l e r t . A drug presumed to in c r e a s e p r o te in synÂ
t h e s i s in th e b r a in and th u s f a c i l i t a t e th e p ro cess of
le a rn in g and memory.
De Novo. Embryonic c u l t u r e .
DNA. D eoxyribonucleic a c id s , th e complex m olecules
of which genes a re composed. They c o n ta in g e n e tic i n f o r Â
mation coded i n sequences of s u b u n its c a l l e d b a se s.
E l e c t r o l y t i c . Produced e l e c t r i c a l charges in
n e u ra l tra n s m is s io n .
17
Engram. The p h y s io lo g ic a l change th a t occurs In
th e nervous system a f t e r an experience and t h i s Is presumed
to be re s p o n s ib le f o r memory. Also known as a memory tra c e .
Enzyme. A b i o lo g ic a l c a t a l y s t - - a substance th a t
a c c e le r a te s a chem ical r e a c tio n .
G l i a . Supporting c e l l s , c e l l s t h a t hold to g e th e r
and keep in p la c e th e neurons; some i n v e s t i g a t o r s claim
t h a t th ey play an im portant r o le i n th e e x c i t a t i o n and
co n duction of nerve im pulses, and as such are r e l a t e d to
le a r n in g .
Hippocampus. A curved nerve t r a c t lo c a te d in th e
f o r e b r a in b e lie v e d to f u n c tio n i n o l f a c t o r y and v i s c e r a l
p ro c e sse s which may p la y a p a r t i n memory sto ra g e p ro cÂ
e sses .
H is to n e . Any o f s e v e ra l p r o te in s complexed, a t
one tim e o r a n o th e r, w ith DNA, whose f u n c tio n i s th a t of
r e g u l a t o r o f DNA.
I n f r a . ( P r e f i x ) , meaning below, beneath, under.
I n t r a c r a n i a l . S itu a te d w ith in th e s k u ll .
In v i t r o . I n j e c t i o n of a r t i f i c i a l c u l t u r e (bacÂ
t e r i a ) i n to th e organism.
18
In v iv o . Something Intro d u ced I n to th e normal
s t a t e o f th e body.
Magnesium Pem oline. A drug t h a t Is a m ild stim u Â
la n t of th e nervous system and Is claim ed to In c re a s e b ra in
b i o s y n t h e s i s .
Messenger RNA. A s in g le s tra n d polymer molecule
c o n ta in in g hundreds of s u b u n its . I t s fu n c tio n i s to convey
g e n e tic in fo rm a tio n from the c e l l nucleus to th e p r o t e i n -
s y n th e s iz in g c e n te r s i n th e c e l l , where, in c o l l a b o r a t io n
w ith ribosom es and t r a n s f e r RNA, i t engages in th e complex
p rocess of p r o te in s y n th e s is .
N eo co rtex . Area of th e b ra in of more re c e n t p y lo -
g e n e tic development. Highly c o r r e la te d w ith in c r e a s in g
im portance of a u d ito ry , t a c t i l e and v is u a l mechanisms—
somatic c o r te x .
N euron. A nerve c e l l . The s t r u c t u r a l and fu n c Â
t i o n a l u n i t of th e nervous system.
N o re p in e p h rin e . A hormone s e c re te d by th e a d re n a l
m edulla t h a t f u n c tio n s as a t r a n s m i t t e r agent a t s y n a p tic
Ju n c tio n s in th e autonomic system.
N u cleu s. (a) A s tr u c t u r e w ith in any c e l l , i n c l u d Â
ing a nerve c e l l . The nucleus c a r r i e s th e g e n e tic inform a-
t i o n of th e c e l l and thus c o n tr o ls th e re p ro d u c tio n of th e
c e l l as w ell as th e chem ical a c t i v i t i e s t h a t ta k e p la c e
elsew here In th e c e l l body. (b) Any p o r tio n o f th e c e n t r a l
nervous system , o th e r th an a c o rte x , In which n e r v e - c e ll
bodies a re c o n c e n tra te d . The b a s a l g a n g lia are n u c le i;
th e hypothalmus and th e thalm us a re each made up o f many
n u c l e i .
P a l r e d - a s s o c l a t e s . M a te ria l used In v e rb a l l e a r n Â
ing, c o n s is t i n g of a l i s t of p a i r s of Items In which one
se rv e s as "stim u lu s" and th e o th e r as " re sp o n se ."
P lyrlbosom e. An a g g re g a tio n of ribosom es.
Puromycln. Produces amino a c id complex th a t subÂ
s t i t u t e s f o r s o lu b le RNA and p re v e n ts a d d itio n o f f u r t h e r
amino a c id s to p o ly p e p tid e s.
R lbam lnol. A drug which Is a s a l t o f s-RNA and
d ie th y la m in o e th a n o l and i s cap ab le o f s tim u la tin g th e
rlbosom al system s In b r a in r e s p o n s ib le f o r p r o te in synÂ
t h e s i s .
Rlbosomal RNA. Found In minute rib o n u c le o -p r o te in
p a r t i c l e s o f nucleus and ribosomes and account f o r about
80 p e rc e n t o f t o t a l c e l l RNA. I t f u n c tio n s as a c o l l a b Â
o r a t o r w ith mRNA to l in k to g e th e r amino a c id s d e liv e re d
by tRNA, th e re b y c r e a tin g p r o te in s .
20
UNA p o ly m era se. A c a t a l y t i c enzyme t h a t s tim u la te s
th e p ro d u ctio n of messenger RNA.
Synapse. The p la c e a t which an axon te rm in a l of
one neuron comes c lo s e to th e d e n d r i ti c zone o f a n o th er
neuron. A c t i v i t y i n th e f i r s t neuron can in flu e n c e a c t i v Â
i t y in th e next one by th e passage o f a t r a n s m i t t e r subÂ
s ta n c e a c ro ss th e s y n a p tic space.
S h o rt-te rm memory. Memory w ith lim ite d c a p a c ity
and s h o rt d u r a tio n .
T ra n s fe r RNA. Account f o r 15-20 p e rc e n t of t o t a l
RNA of th e c e l l . I t fu n c tio n s as a c a r r i e r to a c t i v a t e
amino a c id s d u rin g p r o te in s y n th e s is . I t conveys s p e c i f i c
amino a c id s to s i t e s of p r o te in s y n th e s is . Each of th e s e
amino a c id s has i t s own ty p e o f t r a n s f e r RNA.
T r a n s m itte r s u b s ta n c e . A chem ical t h a t i s r e Â
le a s e d a t th e axon te rm in a l of a neuron and t r a v e l s a c ro ss
th e sy n a p tic space to a f f e c t th e d e n d r i ti c zone o f th e next
neuron. The t r a n s m i t t e r may te n d t o produce a nerve imÂ
p u lse in th e next neuron, i n which case i t i s s a id to be
e x c i t a t o r y . I f i t te n d s to p rev en t an im pulse i n th e next
neuron, i t i s c a l l e d i n h i b i t o r y . Among th e s e t r a n s m i t t e r s ,
two which have been i d e n t i f i e d , are a c e ty lc h o lin e (ACh),
which i s a c h o l i n e r g i c , which r e g u la te s tra n s m is s io n a t th e
21
Ju n c tio n of motor axons and s tr i p e d m uscles; n o re p in e p h rin e ,
which Is a d re n e rg ic , and r e g u la te s tra n sm iss io n a t synapses
In the autonomic system. Also Included I s a c e t y l c h o l i n e s Â
t e r a s e (AChE), an enzyme th a t breaks down ACh a t th e
synapse. This a c tio n I s n e c e ssa ry so t h a t th e sy n a p tic
mechanisms can r e t u r n to a r e s t i n g s t a t e , to be used once
ag ain .
U r a c il. RNA base complementary to DNA adenine.
CHAPTER I I
REVIEW OP THE RELATED LITERATURE
T his c h a p te r d e a ls w ith th e review of th e l i t e r a Â
tu r e im p lic a tin g RNA as th e molecule o f memory. The d i s Â
c u ssio n w i l l cover s tim u la tio n s tu d ie s , s tu d ie s in v o lv in g
p r o te in and n u c le ic i n h i b i t o r s , and s tu d ie s of t r a n s f e r ;
s tu d ie s of th e in g e s tio n of su b stan ces which in c r e a s e th e
p ro d u c tio n of p r o te in and n u c le ic a c id s and t h e i r behavÂ
i o r a l c o r r e l a t e s w ill be reviewed e x te n s iv e ly . A r e s t a t e Â
ment o f th e problem w i l l a lso be in c lu d e d in t h i s c h a p te r
in term s of th e p re se n t re se a rc h , i t s hypotheses and r a Â
t i o n a l e .
S tim u la tio n Tasks and P r o te in S y n th e sis
I t was from an e a r ly study t h a t Hyden (1959)» came
to th e c o n clu sio n th a t RNA and p r o te in s y n th e s is was i n Â
cre a se d fo llo w in g s tim u la tio n to an organism , o r t h a t th e
o p p o site e f f e c t was observed i f d isu se of th e neurons was
m aintained. This g en erated a s e r i e s o f re s e a rc h t h a t has
con tin u ed up to th e p r e s e n t. In a s e r i e s of experim ents,
Hyden and h is a s s o c ia te s ( 1962, 1963* 1964, 1970) t r a in e d
r a t s to walk up a t h i n w ire in o rd e r to o b ta in food, a s Â
suming t h a t t h i s would s tim u la te th e an im a ls' v e s t i b u l a r
22
23
t i s s u e of th e se ex p erim en tal anim als w ith th e same ty p e of
t i s s u e in c o n tr o l r a t s . They found th a t i n both groups of
anim als th e RNA c o n ten t had been r a i s e d , but in th e e x p e r iÂ
mental group o n ly , was th e r e found a change i n th e r e l a t i v e
p ro p o rtio n o f th e two b ases, adenine and u r a c i l . In s im iÂ
l a r experim ents, th ey a ls o dem onstrated base r a t i o changes
in g l i a l RNA im m ediately surrounding D e ite r neurons.
Concerned w ith th e problem of c o n tr o l, Hyden s t i l l
performed a s e r i e s o f experim ents i n which rig h t-h a n d e d
r a t s were t r a i n e d to use t h e i r l e f t paw to reach f o r food.
The advantage of th e s e s tu d ie s was th a t th e c o n tr o l c e l l
m a te r ia l was p re s e n t c o n t r a l a t e r a l l y i n th e c o rte x o f th e
same r a t . When compared w ith the c o n tr o l s id e , s i g n i f i c a n t
in c r e a s e in RNA c o n te n t p e r c e l l o ccu rred on th e le a rn in g
s id e o f th e c o r te x . Based on th e se f in d in g s , th e se r e Â
s e a rc h e rs concluded t h a t an a c u te le a rn in g s i t u a t i o n w ith Â
out p r i o r p reced en t to th e anim al, a c ts as a s tim u la n t
which r e s u l t s i n th e p ro d u c tio n of RNA w ith a l t e r e d base
r a t i o s in th e n u c le i o f d i r e c t l y Involved neurons. From
th e s e f in d in g s , Hyden proposed a mechanism of in fo rm atio n
p ro c e ssin g in th e b r a in i n which n eu ro n al n u c le a r RNA i s
d esig n a te d as th e sensors, c o n s is tin g of frequency modulaÂ
ti o n s which a re re s p o n s ib le f o r th e changes i n base compoÂ
s i t i o n in th e RNA. R ec en tly , he advocated th e involvement
of th e g l i a l c e l l s i n th e a c t i v a t i o n o f RNA which e n te r th e
neuron and a c t as d e r e p re s s io n ag en ts to f r e e a gene o r
24
genes f o r th e p ro d u ctio n of a s p e c i f i c p r o te in which then
in flu e n c e s sy n a p tic c o n d itio n . Prom th e s e b e h a v io ra l s t u Â
d ie s , Hyden et a l . concluded t h a t e a r ly in le a rn in g a DNA-
l i k e RNA i s made i n th e responding nerve c e l l s and th a t
a f t e r th e animal has le a rn e d , a rib o s o m a l-lik e RNA i s p ro Â
duced. Also i t was concluded t h a t s h o rt-te rm memory i s
c h a r a c te r iz e d by th e p ro d u c tio n of an RNA w ith a r e l a t i v e l y
h ig h A d en in e/U racil r a t i o (mRNA), w hile rlbosom al RNA synÂ
t h e s i s c h a r a c te r iz e s th e t r a n s i t i o n from le a rn in g to a
le a rn in g s t a t e .
Commenting on th e se e a r l i e r f in d in g s , G aito s ta t e d :
However, th e base changes appear to be r e l a t e d to senÂ
so ry s tim u la tio n r a t h e r th a n le a r n in g . The sensory
motor a c t i v i t y of b alan cin g on w ire to reach th e p l a t Â
form pro v id es s tim u la tio n f o r th e c e l l s i n th e v e sÂ
t i b u l a r system in th e m edulla but one would expect
t h a t any changes r e p re s e n tin g th e le a rn in g a s p e c ts of
th e a c t i v i t y would be found a t a h ig h e r le v e l i n the
b r a in [1963, p. 4771.
F u rth e r reason f o r sk e p tic ism comes from E ccles:
U n fo rtu n a te ly , t h i s h y p o th e sis ig n o re s th e immense
o r g a n iz a tio n a l s p e c i f i c i t y o f th e nervous system and
in s te a d assumes t h a t th e s p e c i f i c i t y a tta c h e s to th e
frequency p a t t e r n of th e nerve im pulses, and not to
th e l i n e s of communication o f th e nerve f i b e r s . This
h y p o th e sis i s th e r e f o r e u n ac c e p ta b le on n e u ro lo g ic a l
grounds; n e v e r th e le s s Hyden*s experim ents a re o f g re a t
value i n r e l a t i o n to th e orthodox growth th e o ry of
le a rn in g [1963* p. 26o ] .
Classman ( 1969) c o n c e n tra te s h i s c r i t i c i s m mainly
on th e s e grounds:
1. The n a tu re of beh av io rs makes i t d i f f i c u l t to
25
a s c e r t a i n w hether th e r e s u l t s are due to le a r n in g
o r to o th e r a sp e c ts of th e t r a i n i n g p ro c e d u re s.
2. The type of c o n tr o l anim als used i n th e w ire b a l Â
ancing experim ent a re in ad eq u ate to d i f f e r e n t i a t e
between le a r n in g , sensory in p u t, motor a c t i v i t y ,
m o tiv a tio n a l f a c t o r s , s t r e s s , and o th e r psychoÂ
lo g i c a l v a r i a b l e s .
A group o f re s e a r c h e r s from In d ia , Talwar e t a l .
(1966), s tu d ie d th e e f f e c t of s tim u la tio n on B rain RNA.
They s p e c i f i c a l l y i n v e s t i g a t e d th e changes i n th e r a t b r a in
during co nvulsions induced by M etrozole (a c e n t r a l e x c i Â
t a n t ) . These r e s e a r c h e r s a ls o i n v e s tig a te d th e in flu e n c e
of l i g h t s tim u la tio n on th e s y n th e s is of RNA i n th e o c Â
c i p i t a l c o rte x . T h e ir r e s u l t s showed th a t a p o r tio n o f th e
RNA, 10-12 p e r c e n t, undergoes a tu rn o v e r i n response to
s tim u la tio n . S p e c i f i c a l l y , th ey found t h a t moderate s tim Â
u l i ten d to I n c re a s e th e c o n te n t of RNA in th e c e l l s , w hile
in te n s iv e , s u s ta in e d a c t i v i t y or convulsive s t r e s s caused
a d e c re a se i n RNA. They I n te r p r e t e d t h i s to mean t h a t
t h i s d e crease i n RNA r e f l e c t e d th e i n a b i l i t y of th e synÂ
t h e s i s to keep pace w ith u t i l i z a t i o n , where u t i l i z a t i o n
may c o n s is t of e i t h e r :
1. A breakdown of RNA,
2. A tra n s fo rm a tio n of th e type of lin k a g e of RNA
to o th e r c o n s ti t u e n t s , o r
3. A t r a n s f e r o f RNA to o th e r compartments.
A R ussian r e s e a r c h e r , Pevzner ( 1966), summarized
th e r e s u l t s o b tain ed by d i f f e r e n t groups of R ussian in v e s Â
t i g a t o r s concerning th e changes in b r a in RNA metabolism
du rin g e x c i t a t i o n and i n h i b i t i o n of nervous a c t i v i t y .
These r e s u l t s i n d i c a t e t h a t th e r e was an in c re a s e i n RNA
s y n th e s is during e x c i t a t i o n of nerve c e l l s and a p r o l i f e r a Â
t i o n of RNA breakdown during th e i n h i b i t i o n of nerve c e l l s .
Subsequently, Bowman and S tro b e l ( 1 9 6 9)* t r a in e d
r a t s on re p e a te d s p a t i a l r e v e r s a l s f o r w ater rein fo rcem en t
in a Y-maze and found in c re a se d s y n th e s is o f RNA in th e
c e l l n u c le i of th e hippocampus. Unable to f in d a cause
f o r th e s e chem ical changes, th e y su g g ested t h a t th e se may
be due to th e le a r n in g event, o r to s p e c ia l s t r e s s e s and
em otional and m o tiv a tio n a l e f f e c t s of le a rn in g which are
re s p o n s ib le f o r t r i g g e r i n g such chem ical re sp o n ses; i t i s
a ls o p o s s ib le t h a t th e changes in RNA a re due to nonÂ
s p e c i f i c s tim u li o r to th e a c t i v i t y a s s o c ia te d w ith th e
t r a i n i n g e x p e rie n c e . Also Zemp e t a l . (1970), found i n Â
crea se d RNA s y n th e s is in th e mouse su b c o rte x during a v o id Â
ance c o n d itio n in g . These r e s e a r c h e r s were unable to exÂ
p l a i n th e source of t h i s in c r e a s e w hether i t was due to
e a r l y s te p s of memory c o n s o lid a tio n o r re p re s e n te d sim ple
m aintenance and re co v ery p ro c e sse s a s s o c ia te d w ith n eu ro n al
fu n c tio n in g . Supportive evidence f o r t h i s second i n t e r Â
27
p r e t a t i o n comes from Appel et a l . ( 1 9 6 7)* who re p o rte d
In c re a s e d polysome ag g re g a tio n In th e v i s u a l c o rte x of
r a t s given 15 m inutes of exposure to l i g h t follow ing th re e
days of confinem ent i n th e dark. No le a rn in g was in v o lv ed ,
and t h i s i n c r e a s e in RNA i s s tr o n g ly su g g estin g th e mainÂ
ten an ce f u n c tio n o f t h i s m olecule.
And f i n a l l y , Shashua ( 1968, 1970), found a l t e r a Â
ti o n s i n c r e a s e in u r i d i n e / c y t i d i n e r a t i o s in RNA of g o ld Â
f i s h le a rn in g to swim w ith a f l o a t a tta c h e d to t h e i r u n d erÂ
s id e . These in c r e a s e s were not observed in c o n tr o l groups
g iv en v a rio u s tre a tm e n ts such as puromycin i n j e c t i o n s ,
swimming in a " w h irlp o o l," swimming w ith a f l o a t too la rg e
to manage, o r KCL-induced co n v u lsio n , e tc .
Shahua th e n s ta t e d :
The fin d in g s in t h i s paper suggest th a t changes i n
RNA s y n th e s is in g o ld fis h b ra in d u rin g th e a c q u i s i Â
t i o n of a new b e h a v io ra l p a t t e r n , a re not s p e c i f i c
w ith r e s p e c t to th e in fo rm a tio n c o n te n t being s to re d
but may be re q u ire d f o r c o n s o lid a tio n s te p of i n f o r Â
m ation s to r a g e [1970, p. 1 6 6].
P r o te in and N ucleic I n h i b i t o r s
O ther i n v e s t i g a t o r s have approached t h i s problem by
a d m in is te rin g p r o t e i n i n h i b i t o r s to anim als and observing
t h e i r b e h a v io ra l e f f e c t s .
F le x n e r e t a l . ( 1963, 1967)* In a few o f many s t u Â
d ie s w ith drugs and t h e i r e f f e c t s on memory, used s e v e ra l
i n h i b i t o r s of p r o t e i n s y n th e s is , such as puromycin and
acetoxycyclohexim ide. These r e s e a r c h e r s in j e c t e d puromycin
i n to th e tem poral a re a s of th e b ra in of mice a t v ario u s
tim es a f t e r they had le a rn e d an avoidance response in a Y-
maze. The mice were th en t e s t e d f o r r e t e n t io n of th e
avoidance response th re e days fo llo w in g th e puromycin i n Â
j e c t i o n . When puromycin was i n j e c t e d s ix days o r more
follow ing i n i t i a l le a rn in g , th e mice showed no r e t e n t io n of
th e avoidance resp o n se. These re s e a r c h e r s concluded th a t
puromycin I n t e r f e r e d w ith p r o te in s y n th e s is in c e r t a i n
b ra in reg io n s and blocked s h o rt- te rm memory, but not longÂ
term memory. Also, AXM caused i n h i b i t i o n of p r o te in synÂ
t h e s i s in a l l c e r e b ra l a re a s e q u iv a le n t to t h a t produced
by puromycin. However, i n c o n t r a s t to puromycin, the
heximide had no e f f e c t on e i t h e r re c e n t o r long-term r e Â
t e n t i o n ta s k s . Furtherm ore, AXM p r o te c te d memory a g a in st
th e d e s tr u c t i v e e f f e c t s of puromycin, alth o u g h m ixture of
th e two drugs produced profound i n h i b i t i o n of p r o te in synÂ
t h e s i s .
Working w ith g o ld f is h , Agranoff e t a l . ( 1966,
1967), in j e c t e d puromycin i n t r a c r a n i a l l y in to th e f i s h a t
v ario u s tim es fo llow ing th e le a r n in g of an avoidance ta s k
in a s h u t t l e tan k . The g r e a t e s t d e f i c i t s were shown when
puromycin was im m ediately i n j e c t e d a f t e r le a rn in g , and
l e s s e r d e f i c i t s were found when puromycin was ad m in istered
30 minutes a f t e r le a rn in g of th e avoidance ta s k : th e r e
were no d e f i c i t s a t a l l i f th e puromycin was ad m in istered
as l a t e as one hour a f t e r th e le a rn in g had ta k e n p la c e .
F urtherm ore, th ey found th a t puromycin i n j e c t e d p r i o r to
i n i t i a l le a rn in g d id not im p air avoidance le a rn in g , but
i t did im p air th e subsequent r e t e n t i o n of t h a t ta s k . The
d i f f e r e n c e in th e e f f e c t s of puromycin and AXM were a t Â
t r i b u t e d by F le x n e r e t a l . ( 1967) to th e f a c t t h a t th e
hexim ides suppress only p r o te in s y n th e s is , w hile puromycin
su p p resses the s y n th e s is of both messenger RNA and p ro Â
t e i n s . I f t h i s h y p o th e sis i s c o r r e c t , a drug t h a t i n h i b i t s
RNA s y n th e s is should produce a d e f i c i t in memory. A number
of i n v e s t i g a t o r s , however, have f a i l e d to f in d any d e f i c i t
i n th e r e t e n t i o n e i t h e r o f a p a ssiv e avoidance response o r
a p o s it i o n d is c r im in a tio n in mice and r a t s a f t e r i n j e c t i o n
o f Actinomycin D, a drug which su p p resses RNA s y n th e s is
(Appel, 1965; Cohen e t a l . , 1966; Goldsmith, 1967). ReÂ
c e n tly , A granoff e t a l . ( 1967) did f in d a d e f i c i t in r e Â
t e n t i o n o f an a c t i v e avoidance response i n g o ld f is h by
t e s t i n g th e f i s h th re e days a f t e r o r i g i n a l le a rn in g and
i n j e c t i o n . They i n t e r p r e t e d th e f a i l u r e by p rev io u s i n Â
v e s ti g a t o r s as being due to a r e s i d u a l s h o rt-te rm memory.
Commenting on th e e f f e c t s of a n t i b i o t i c s , Appel
e t a l . s ta t e d :
These s tu d ie s im p lic a te de novo p r o te in s y n th e s is as
v i t a l to in fo rm a tio n p ro c e ssin g by d em o n stratin g t h a t
a n t i b i o t i c s which i n h i b i t p r o te in s y n th e s is a l t e r
s p e c i f i c b e h a v io r. Such s tu d ie s a re always s u b je c t
to th e u n c e r t a i n t y of w hether th e drugs produce t h e i r
30
e f f e c t s by t h e i r s t a t e d a c tio n on p r o te in s y n th e s is ,
o r by some o th e r e f f e c t r e l a t e d e i t h e r to o th e r a c Â
tio n s of th e drug o r to secondary r e p e rc u s s io n s of
th e a l t e r e d p r o te in s y n th e s is [1970, p. 9 2].
R ecently F le x n e r and F lex n er ( 1968) have shown t h a t
b r a in i n j e c t i o n s of s a li n e w i l l reduce th e d e f i c i t i n memÂ
ory produced by puromycin, a fin d in g which in d i c a t e s th a t
puromycin blocks th e e x p re ssio n of memory r a t h e r th an i t s
fo rm atio n . Also, more re c e n t s tu d ie s (Barondes & Cohen,
1968) suggest t h a t cyclohexim ides, p o te n t i n h i b i t o r of
b ra in p r o te in s y n th e s is , can prev en t th e form ation o f lo n g Â
term memory w ithout a f f e c t i n g s h o rt-te rm memory. Along
a n o th er l i n e of i n v e s t i g a t i o n , Cohen and Barondes ( 1967)
have p re se n te d evidence to show th a t th e e f f e c t s of pu ro Â
mycin on memory may be due to sm all " o c c u lt" s e iz u r e s .
E v id e n tly , puromycin makes sm all h o le s in th e b ra in t i s s u e
and th e r e f o r e i t s e f f e c t s on p r o te in s y n th e s is a re s e r i o u s Â
ly confounded w ith th e s e s e iz u re s i n re g a rd to memory.
F u rth e r evidence su p p o rtin g t h i s in fo rm a tio n ,
comes from a r e c e n t, w e ll-d e sig n e d study i n which Nakajlma
( 1 9 6 9) compared th r e e groups of r a t s . In one group, a c Â
tinom ycin D was I n je c t e d in to th e a n t e r i o r n eo co rtex .
When t e s t e d on a p o s it i o n d is c r im in a tio n ta s k in a T-maze,
th e group w ith th e drug i n j e c t e d i n to th e hippocampus
showed d e f i c i t s i n both le a r n in g and r e t e n t io n ; c o r t i c a l
I n je c t io n o f th e same drug d id not g iv e r i s e to such
d e f i c i t s in th e second group of anim als; whereas le s io n s
31
In the hippocampus produced a d e f i c i t i n le a rn in g but not
r e t e n t i o n . H is to lo g ic a l exam inations showed t h a t , a t th e
tim e of th e b e h a v io ra l t e s t , th e s y n th e s is o f a new RNA
was stopped, and th a t RNA-dependent p r o t e i n s y n th e s is was
a l s o suppressed. The b e h a v io ra l c o n d itio n s under th e se
c o n d itio n s were s im ila r to th e r e s u l t s o b ta in e d w ith pu ro Â
mycin (F lexner e t a l . , 1963) and A X M (Barondes & Cohen,
I 96J ) , and agree w ith th e fin d in g s by Agranoff et a l .
( 1967) u sin g actinom ycin D on th e g o ld f is h . However, in
h i s i n t e r p r e t a t i o n of the r e s u l t s , Nakajima a t t r i b u t e d the
d e f i c i t s , not to th e su p p re ssio n of macrom olecular synÂ
t h e s i s , but to th e spike d isc h a rg e s in th e hippocampal
re g io n a f t e r drug i n j e c t i o n . In h is c o n c lu sio n , Nakajima
s t a t e d :
I t now seems n e cessary to re-exam ine th e r e s u l t s of
the memory experim ents i n which e p ile p to g e n ic a n t i Â
b i o t i c s were used [ 1969* P* ^ 6 l] .
In a re c e n t a r t i c l e , based on v a rio u s experim ents,
Barondes summarized h is co n clu sio n s as fo llo w s:
Learning and s h o rt-te rm memory ( f o r a t l e a s t th re e
hours a f t e r t r a i n i n g in th e s i t u a t i o n s tu d ie d ) are
a p p a re n tly Independent of c e r e b r a l p r o te in s y n th e s is .
Presumably some o th e r mechanism i s re s p o n s ib le f o r
memory sto ra g e f o r t h i s p e rio d . ( 2 ) I t i s d i f f i c u l t
to i n t e r p r e t th e e f f e c t s o f puromycin on memory sin c e
t h i s agent not only i n h i b i t s c e r e b r a l p r o te in s y n th e s is
but a ls o in c r e a s e s c e r e b ra l i r r i t a b i l i t y and th e p ro Â
p e n s ity to o c c u lt s e iz u re s which could I n t e r f e r e w ith
memory. ( 3 ) Prolonged t r a i n i n g i n a sim ple ta s k may
obscure th e e f f e c t s of i n h i b i t i o n t o c e r e b r a l p r o te in
s y n th e s is on memory, presumably because th e sm all
amount of r e s i d u a l p r o te in s y n th e s iz in g c a p a c ity may
be s u f f i c i e n t to m ediate memory s to ra g e under t h i s
32
c o n d itio n . (4) A cetoxycyclohexim lde, an agent which
does not a p p a re n tly i n t e r f e r e w ith i n t r a c e r e b r a l e l e c Â
t r i c a l a c t i v i t y , im p airs "long term" memory s to r a g e .
However, th e p o s s i b i l i t y t h a t th e drug produces amÂ
n e s ia by some o th e r mechanism cannot be excluded.
(5) The p r o te in s y n th e s is a p p a re n tly re q u ire d fo r "long
term" memory s to ra g e occurs d u rin g le a rn in g a n d /o r
w ith in minutes a f t e r le a rn in g . (6) Since th e amnesic
e f f e c t of acetoxycyclohexim lde i s not observed u n t i l
s ix hours a f t e r t r a i n i n g , th e " sh o rt term" mechanism
which i s a p p a re n tly independent o f p r o te in s y n th e s is
may su rv iv e u n t i l t h i s time and te m p o ra rily obscures
th e e f f e c t of th e i n h i b i t o r on th e long term" p ro c Â
ess [1970, p. 32].
And now, S e ro ta et a l . (1972), t e s t e d th e p o s s i b i l Â
i t y th a t a d re n e rg ic s tim u la n ts might provide p r o te c tio n
from the amnesia. The drug Metaraminol was used as i t
a c ts as a f a l s e t r a n s m i t t e r and a pow erful agent f o r th e
displacem ent of n o re p in e p h rin e i n h i b i t i n g i t s u ptake.
Metaraminol was given e i t h e r b e fo re o r a f t e r t r a i n i n g and
b efo re t e s t i n g to p rev en t th e drug D-amphetamine from d e Â
s tro y in g n o re p in e p h rin e and to p rev en t amnesia. These
r e s e a rc h e rs claim ed t h a t t h e i r o b s e rv a tio n s i n d i c a t e t h a t
agents which in c r e a s e th e a v a i l a b i l i t y of n o re p in e p h rin e
a t th e tim e of t r a i n i n g , th e f i r s t two hours during conÂ
s o l i d a t i o n , o r a t th e time o f t e s t i n g , p r o te c t a g a in s t
t r a n s i e n t amnesia. In p re s e n tin g t h e i r co n clu sio n s S e ro ta
et a l . s ta t e d :
These fin d in g s suggest t h a t acetoxycyclohexim lde
(AXM) produces i t s e f f e c t by red u cin g th e amount of
a v a ila b le n o re p in e p h rin e , a c o n s id e ra b le d e p a rtu re
from an e a r l i e r view based on assumed r o le s in memory
of messenger RNA p r o te in [1972, p. 342].
T ra n s fe r S tu d ies
The o r i g i n a l s tu d ie s in v o lv in g " t r a n s f e r of a c Â
q u ire d b e h a v io ra l te n d e n c ie s" was conducted by McConnell
e t a l . (1961, 1962). In th e s e s tu d ie s , p la n a ria n s were
s u b je c te d to c l a s s i c a l c o n d itio n in g t r a i n i n g u sin g l i g h t
as CS and e l e c t r i c shock as UCS. A fte r t r a i n i n g , th e s e
p la n a r ia n s were chopped in p ie c e s and fed to u n tra in e d ,
c a n n i b a l i s t i c flatw orm s. The flatw orm s were th en given
c o n d itio n in g t r i a l s . The response r a t e s to l i g h t (CS) of
th e s e exp erim en tal anim als were from th e f i r s t s e t of
t r a i n i n g t r i a l s s i g n i f i c a n t l y g r e a t e r th a n th e response
r a t e s of c o n tr o l c a n n ib a ls fed th e bodies of u n tra in e d
v ic tim s . Also, i t was McConnell who was f i r s t to show
t h a t a crude RNA m ixture e x tr a c te d from th e bodies of conÂ
d itio n e d p la n a ria n s and i n j e c t e d in to th e bodies o f unÂ
t r a in e d flatw orm s a p p a re n tly caused an in c r e a s e i n response
r a t e s i n th e exp erim en tal anim als, as compared w ith th e
b eh av io r of s e v e ra l groups of c o n tr o l anim als i n j e c t e d
w ith a crude RNA m ixture e x tr a c te d from th e bodies of
v a rio u s groups of u n tra in e d p la n a r ia n s . O ther la b o r a to r ie s
r e p l i c a t e d th e s e r e s u l t s and found n e g a tiv e r e s u l t s , among
them Brown (1964, 1967) and Crawford ( 1967). ------------------
Cornig and R iccio (1970) p re se n te d an ex h au stiv e
review of th e fin d in g s r e l a t e d to th e p l a n a r i a c o n tro v e rsy .
The main c r i t i c i s m a g a in s t th e t r a n s f e r s tu d ie s i s t h a t th e
34
t r a n s f e r f a c t o r causes in c re a se d s e n s i t i v i t y to s ti m u l i o r
in c re a se d a c t i v i t y (Glassman, 1969)•
Experiments su g g estin g a s im i l a r " t r a n s f e r e f f e c t "
i n b e h a v io ra l ta s k s in v o lv in g h ig h e r organism s, such as
r a t s and h am sters, were performed by many r e s e a r c h e r s in
d i f f e r e n t p a r ts of th e world. Byrne (1970) p re s e n te d a
d e t a i l e d review of th e se s tu d ie s g iv in g a v a s t amount of
in fo rm a tio n . The evidence i s much i n fav o r of th e p o s i t i v e
e f f e c t s of th e se t r a n s f e r s tu d ie s . Weiss (1970) remarked
t h a t th e range of c o n tr a d ic to ry r e s u l t s and th e c o n s is te n t
d i f f i c u l t y ev id en t in th e atte m p ts a t r e p l i c a t i o n i s i n Â
d i c a t i v e o f th e c u rre n t la c k of u n d erstan d in g o f th e u n d erÂ
ly in g mechanisms which a re re s p o n s ib le f o r the t r a n s f e r
phenomenon.
RNA as an Inducer of Learning
The h y p o th e sis t h a t th e use of a su b stan ce o r an
agent to in c r e a s e p r o te in and n u c le ic s y n th e s is r e s u l t s
i n a f a c i l i t a t i o n o f memory c o n s o lid a tio n i s b a s ic to th e
s tu d ie s in v o lv in g th e use o f drugs to improve le a r n in g .
This assum ption has led i n v e s t i g a t o r s to s p e c u la te t h a t
inducing lo c a l i z e d in c r e a s e s in p r o te in s y n th e s is w ith
drugs may have th e e f f e c t of in c r e a s in g memory c o n s o lid a Â
t i o n i n th e organism , th e r e s u l t of which co u ld o f f e r
g r e a t s ig n if ic a n c e f o r improving c o g n itiv e p ro c e sse s i n
humans.
35
Among th e su b stan ces used to in c r e a s e RNA s y n th e s is
i n th e b ra in i s th e su bstance o f magnesium pem oline. The
use of magnesium pemoline i n s e v e ra l animal l a b o r a t o r i e s
has r e s u l t e d i n p o s it i v e evidence f o r th e a b i l i t y o f t h i s
su bstance to a c t as a f a c i l i t a t o r in avoidance c o n d itio n in g
as w ell as a m ediator f o r in c r e a s in g th e amount of RNA in
th e b ra in (Glasky & Simon, 1 9 6 6; Simon & Glasky, 1968;
P lo tn ik o f f , 1966a , b , c ; P lo tn ik o ff & Meekma, 1967). F u rth e r
su p p o rtin g evidence comes from s tu d ie s in v o lv in g th e i n Â
j e c t i o n of RNA, which r e s u l t e d i n f a c i l i t a t i o n of le a rn in g
and d elay s in e x t i n c t i o n o f an avoidance response (Cook
et a l . , 1966; Wagner, 1966; S ie g a l, 1967* 1968). However,
c o n f l i c t i n g evidence has been re p o rte d from s e v e ra l s tu d ie s
w ith magnesium pemoline and RNA p ro d u ctio n (Cain, 1967;
M orris e t a l . , 1967; S te in & J e l l i n , 1967; Frey & P i l i d o r a ,
1967; Beach & Kimble, 1967; S ie g a l e t a l . , 1967; S te in
et a l . , 1 9 6 8). The amount of r e s e a r c h in v o lv in g RNA i n Â
g e s tio n and anim al b e h a v io ra l s tu d ie s r e l a t e d to le a r n in g
and memory i s v a s t; however, i n th e a r e a of RNA i n human
le a rn in g and memory, re se a rc h has not been so abundant.
S tu d ies w ith th e Aged
To v a l i d a t e th e p o s s i b i l i t y t h a t in c re a se d RNA i n Â
g e s tio n can enhance memory in th e aged, a s e r i e s o f in v e s Â
t i g a t i o n s has been c a r r i e d out by Cameron and h is a s s o c i Â
a te s (Cameron, 1958; Cameron & Solyom, 1961; Cameron,
36
Solyom & Beach, 1 9 6 1 ; Cameron, Sved, Solyom, Winrib &
B a rlk , 1963; Cameron, K rai, Solyom, Sved, W alnrib, B eaulieu
& Enesco, 1 9 6 6). In g e n e ra l, th ey found p o s i t i v e r e s u l t s .
In an e a r ly stu d y , Cameron (1958) a d m in iste re d RNA c ap su les
and RNA i n j e c t i o n in tra v e n o u s ly to 23 aged p a t i e n t s w ith
s e n i l e dem entia. Using counting t e s t s as an a s s e s s o r of
memory, he re p o rte d t h a t a l l Ss showed improvement w hile
th e more s e v e re ly d e t e r i o r a t e d p a t i e n t s showed th e g r e a t e s t
amount of improvement. In subsequent s tu d ie s (Cameron &
Solyom, 1961; Cameron, Solyom & Beach, 1 9 6 1) w ith improved
methodology and s t i l l u sin g g e r i a t r i c p a t i e n t s , th e study
was re p e a te d d iv id in g th e s u b je c ts in to two groups. Group
I , 37 Ss, re c e iv e d y e a s t o r a l l y o r in tra v e n o u s ly ; Group I I ,
43 Ss, re c e iv e d y e a s t RNA o r a placebo o r a l l y in a dou b leÂ
b lin d c o n d itio n . E v a lu a tio n of Group I Ss was a sse sse d on
th e b a s is of t h e i r perform ance on a co unting t e s t , th e
W echsler Memory S cale, and on in fo rm a tio n su p p lie d by th e
p a t i e n t and h is r e l a t i v e s .
These r e s e a r c h e r s r e p o rte d o v e r a l l improvement i n
23 of th e 37 Ss i n Group I . Improvement i n r e t e n t i o n span,
as measured by th e counting t e s t u s u a lly was noted a f t e r
th e f i r s t th r e e weeks of tre a tm e n t. They a ls o re p o rte d
g e n e ra l improvement i n perform ance on th e Wechsler Scale
but f a i l e d to p ro v id e d a ta f o r th e i n d iv id u a l s u b te s ts .
G re a te r improvement was observed i n th e a r t e r i o s c l e r o t i c
th a n In th e s e n il e dem entia p a t i e n t s . The re s e a rc h e rs
a ls o re p o rte d t h a t a l e s s e r amount of RNA was n e c e ssa ry to
achieve improvement when RNA was ad m in iste re d in tra v e n o u sly .
Caution i s urged in th e v a l i d i t y of th e s e r e s u l t s from
Group I s in c e a placebo c o n tr o l group was not employed.
R e su lts in Group I I , o r th o se p a t i e n t s t r e a t e d w ith RNA,
were comparable to th o se o b ta in e d in Group I , but improveÂ
ment was somewhat l e s s . S p e c i f ic a l ly , no s i g n i f i c a n t imÂ
provement was observed in performance of p a t i e n t s on th e
Wechsler Memory S cale, but a s i g n i f i c a n t improvement was
observed i n perform ance of p a t i e n t s on th e co unting t e s t
f o r th o se a r t e r i o s c l e r o t i c p a t i e n t s t r e a t e d w ith RNA.
Improvement of s e n i l e dem entia p a t i e n t s was observed only
i n Group I . In g e n e r a l, th e se r e s e a r c h e r s found th e g r e a t Â
e s t improvement i n perform ance i n th o se in s ta n c e s in which
memory d e t e r i o r a t i o n was mild to moderate and l e a s t In
th o se cases i n which memory d e t e r i o r a t i o n was advanced.
These fin d in g s c o n t r a d i c t th e Cameron (1958) fin d in g s .
Using a more c o n c e n tra te d s o lu tio n of y e a s t RNA,
Cameron, Solyom, Sved, and Wainrib ( 1963) found improvement
i n memory fo llo w in g RNA in tra v e n o u s I n j e c t i o n to 16 p r e -
s e n il e and s e n il e p a t i e n t s on t e s t - r e t e s t measures of th e
Wechsler Memory S cale and on th e counting t e s t . This study
s u ffe re d from many m ethodological weaknesses among which
i s th e la c k of i n t e r n a l v a l i d i t y (Campbell & S ta n le y ,
1966), s in c e a c o n tro l placebo group was not employed. In
a s im i l a r stu d y , w ith a la r g e r sample o f Ss, Cameron, Sved,
Solyom, Wainrib and B arik (1963)* re p o rte d improvement in
memory, but s t i l l w ithout a placebo c o n tr o l group.
In r e fe r e n c e t o th e se s tu d i e s , G aito ( 1 9 6 3) s tre s s e d
th e p o in t th a t th e s tu d ie s made by Cameron et a l . , may be
more r e le v a n t to n u t r i t i o n a l needs than to memory. Also
th e in g e s te d RNA may in c re a s e th e pool from which RNA conÂ
s t i t u e n t s are drawn and th ereb y ten d to improve th e o v e r a ll
c o n d itio n of o ld e r in d iv id u a ls who have a d e f i c i e n t amount
of RNA a v a i l a b l e (Hyden, 1961). Continuing h is d is c u s s io n
of th e s e s tu d i e s , Gaito s ta te d :
Furtherm ore, th e re a re p o s s ib le m ethodological d e f i Â
c ie n c ie s i n th e se s tu d ie s . In the Cameron s tu d i e s ,
t h e r e i s no i n d i c a t i o n th a t measures were ta k e n to
p re v e n t b ia s from a f f e c t i n g th e improvement r a t i n g s of
each of th e s u b je c ts in th e experim ents. The r a t i n g s
were based on performance of s e v e ra l ta s k s , su p p le Â
mented by in fo rm a tio n su p p lie d by th e p a t i e n t and h i s
r e l a t i v e s . As i s w e ll known, r e s u l t s of drug s tu d ie s
such as t h i s can be g r e a t l y a f f e c te d by th e a t t i t u d e s
and e x p e c ta tio n s o f th e p a t i e n t and h o s p i t a l p e rso n Â
n e l i f d o u b le -b lin d procedures are not employed.
However, i n one o f th e two experim ents re p o rte d by
Cameron and Solyom ( 1961), d o u b le -b lin d procedures
were used and some improvement re p o rte d [1963* p. ^76].
In g e n e r a l, th e major c r i t i c i s m s a g a in s t th e CamÂ
eron e t a l . s tu d ie s i s th a t th ey d id not employ adequate
ex p erim en tal c o n tr o ls and i n many in s ta n c e s , th e s t a t i s t i Â
c a l a n a ly s is of th e d a ta was incom plete.
N egative r e s u l t s of th e e f f e c t s o f RNA came from a
w ell c o n t r o ll e d d o u b le -b lin d stu d y by Nodine, Shulkin,
39
S lap, Levine, and F re ib e rg ( 1967). F ifty - o n e p a t i e n t s
diagnosed as s e n i l e o r a r t e r i o s c l e r o t i c b r a in d ise a se d were
randomly a ssig n ed t o RNA, a placeb o , and to an exp erim en tal
a g en t. P retre a tm e n t b a s e lin e measures were o b ta in e d f o r
counting p u r s u it r o t o r , ta p in g speed and r e a c t i o n time
t e s t . P a tie n ts were th en g iv en o r a l l y 10 mg of a s y n th e tic
RNA, an I n e r t p laceb o , or U -I 98O (1500 mg) f o r fo u r weeks.
R e su lts were e v a lu a te d by r e p e a tin g th e t e s t s th e
f i r s t , second, f o u r th , and s i x t h weeks from th e i n i t i a t i o n
of th e tre a tm e n t. No s i g n i f i c a n t d if f e r e n c e s were found
In perform ance between th e PINA, placebo o r experim ental
agent groups. On the c o n tr a ry , th e p la c e b o - tr e a te d groups
showed n o tic e a b le improvement In i n t e l l e c t u a l f u n c tio n in g .
These r e s e a r c h e r s a t t r i b u t e t h i s improvement to th e a t t e n Â
t i o n t h a t th e p a t i e n t s re c e iv e d during th e study, and to
th e p r a c t i c e o f th e e f f e c t s i n th e t e s t s i t u a t i o n s . I t
should be noted t h a t Cameron claim ed th e g r e a t e s t improveÂ
ment i n RNA f o r th o se p a t i e n t s who had been given the.
s te r o i d in tra v e n o u s ly , w hile in th e Nodine et a l . stu d y ,
RNA was a d m in iste re d o r a l l y .
In a much b e t t e r c o n tr o lle d stu d y , T a lla n d , Mendel-
son, Koz and Aaron (1965) a d m in iste re d th e drug T riap t o 24
s e v e re ly d e t e r i o r a t e d p a t i e n t s and f a i l e d to f in d any RNA
r e l a t e d increm ents I n memory f u n c tio n in g . Placebo c o n tro ls
were used i n a d o u b le -b lin d , cro sso v e r ty p e o f d e sig n . In
40
one group, each p a t i e n t was g iv en T riap f o r th e f i r s t two
weeks and a placebo d u rin g th e second two weeks. A second
group re c e iv e d th e se tre a tm e n ts i n th e re v e rs e o rd e r. Each
S _ was t e s t e d s ix tim es on p a r a l l e l s e ts of f iv e p sy c h o lo g iÂ
c a l t e s t s . They found t h a t n e i t h e r th e tre a tm e n t nor th e
p r a c t i c e helped to r e s t o r e l o s t c a p a c i t i e s .
According to Glasky and Simon (196 6 ), magnesium
pemoline f a c i l i t a t e d th e s y n th e s is of n u c le ic a c id s in th e
b r a in of r a t s . Thus, th ey arg u e, magnesium pemoline may
h e lp to c l a r i f y th e r e l a t i o n s h i p between RNA s y n th e s is o r
p r o te in s y n th e s is , o r b oth, and th e p ro c e sse s u n d erly in g
le a rn in g and memory. But, i n s p i t e of th e s e claim s, E is -
d o r f e r , Conner, and Wilke ( 1 9 6 8) o r a l l y a d m in iste re d magÂ
nesium pemoline to 29 male p a t i e n t s from 55 to J Q y e a rs
old and f a i l e d to d e te c t a le a r n in g o r memory enhancement.
S tu d ie s w ith Normal S u b jects
In a study w ith 39 a d u lt men, Smith ( 1 9 6 7) adminÂ
i s t e r e d o r a l doses of 25 mg and 37.5 mg of magnesium pemoÂ
l i n e and a placebo to th e se Ss on a d o u b le -b lin d b a s is .
E v a lu a tio n o f le a rn in g and r e t e n t i o n in clu d ed s e r i a l v e rb a l
le a rn in g , motor perform ance, and c l a s s i c a l c o n d itio n in g .
R e s u lts in d ic a te d t h a t magnesium pemoline does not f a c i l i Â
t a t e le a rn in g , memory, o r perform ance on normal a d u lt men.
"In f a c t , th e only s t a t i s t i c a l l y s i g n i f i c a n t e f f e c t s i n d i -
41
c a te t h a t th e h e a v ie r dose Is d e le te r io u s f o r v e rb a l and
motor le a rn in g [p. 6 0 5] ."
F u rth e r n e g a tiv e r e s u l t s come from a stu d y w ith 30
male u n i v e r s i t y s tu d e n ts o f above average I n t e l l i g e n c e .
Burns, House, Fensch and M ille r ( 1967) used a d o u b le -b lin d
procedure and o r a l l y a d m in iste re d 6 . 2 5, 12.5 and 25 mg of
magnesium pem oline, a p laceb o , and dextroamphetamine to
f iv e groups of s u b je c ts randomly assig n ed to each group.
Two ta s k s were used, a s im p le -re a c tio n -tim e t a s k and a
c o m p le x -re a c tio n -tim e ta s k . R e su lts in d ic a te d t h a t magneÂ
sium pemoline does not enhance human le a rn in g . Burns, e t
a l . d id observe t h a t th e le a rn in g r a t e under lower doses
of magnesium pemoline looked p r o g re s s iv e ly more l i k e p l a c e Â
bo s c o re s . However, Gelfand et a l . ( 1967) gave 100 mg of
magnesium pemoline o r a l l y to 21 young male f a tig u e d subÂ
j e c t s . When compared w ith a placebo group in a d o u b leÂ
b lin d d esig n , th e y found t h a t magnesium pemoline (100 mg)
f a c i l i t a t e d p ro b le m -so lv in g perform ance on r e p e t i t i o u s
a r ith m e tic problems f o r a p erio d of f iv e h o u rs. These r e Â
s e a rc h e rs a t t r i b u t e d th e improvement i n p ro b lem -solving
a b i l i t i e s to th e s tim u la tin g e f f e c t s o f magnesium pemoline
in c o u n te ra c tin g f a t i g u e .
In a n o th e r study, T a llan d and McGuire ( 1 9 6 7) o r a l l y
ad m in iste re d 25 mg o f th e drug C y lert (pemoline w ith magÂ
nesium hydroxide) and a la c to s e placebo to 20 male volunÂ
t e e r s . Using maze le a r n in g , n a r r a t i v e t e x t , and f ig u r e
drawings as measures of perform ance, th e s e s u b je c ts were
t e s t e d f o r le a rn in g , r e c o g n itio n , and r e c a l l . R e su lts i n Â
d ic a te d no improvement a t t r i b u t a b l e to C y le rt on new l e a r n Â
ing of th e se d i f f e r e n t ta s k s . However, r e le a r n in g of th e
maze re q u ire d fewer t r i a l s a f t e r m edication w ith C y le rt
th an d id th e r e le a r n in g under a l t e r n a t e tre a tm e n ts . Also,
b e t t e r r e t e n t i o n of th e p a t t e r n was m aintained over a
w eek's p e rio d f o r th o se t r e a t e d w ith C y le rt. The e f f e c t
was a t t r i b u t e d to th e o v e r a l l stim u la n t or a n t i - f a t i g u e
e f f e c t of C y le rt.
Before summarizing and concluding th e r e s u l t s of
s tu d ie s made on th e use of a su b stan ce o r an agent to i n Â
c re a se p r o te in and n u c le ic s y n th e s is f o r th e purpose of
f a c i l i t a t i n g le a rn in g and memory c o n s o lid a tio n , i t i s
n e c e ssa ry to review th e r e s u l t s found in s tu d i e s u sin g a
new chem ical su b stan ce Ribaminol (hydroxytriethylammonium
r i b o n u c l e a t e ) . Simon, Glasky, and Luebben ( 1968), found
t h a t t h i s su b stan ce i s capable of enhancing b r a in p r o te in
s y n th e s is in r a t s when a d m in iste re d i n vivo o r in v i t r o .
Ribaminol S tu d ies
In a r e l a t e d stu d y which was concerned w ith th e
e f f e c t s of Ribaminol on c o n d itio n e d avoidance le a rn in g ,
Glasky e t a l . ( 1968) used male and female r a t s between th e
ages o f two and tw elve months in an avoidance c o n d itio n in g
ta s k w ith e l e c t r i c shock (UCS) u sin g a T-shaped w ater maze
as a le a r n in g ta s k . Using d i f f e r e n t doses of Ribaminol and
a s a l i n e placebo as tre a tm e n ts f o r each anim al, th e r a t s
were t e s t e d on th e avoidance and th e maze le a rn in g ta s k s .
R e su lts in d ic a te d t h a t Ribaminol was e f f e c t i v e in i n c r e a s Â
ing th e r a t e of a c q u i s it i o n o f th e avoidance le a rn in g in
th e jump-out t e s t and in th e w ater T-maze f o r both male
and fem ale r a t s . They a lso re p o rte d th e enhancing e f f e c t s
of th e drug in r e t e n t i o n le a rn in g . These r e s e a r c h e r s a t Â
t r i b u t e d th e enhancing e f f e c t s to th e s p e c i f i c chem ical
n a tu re of th e compound which, th ey claim ed, f a c i l i t a t e s
p r o te in s y n th e s is in th e b r a in . I t must be noted t h a t
th e s e r e s e a r c h e r s did not in d i c a t e c l e a r l y t h e i r method of
assignm ent of s u b je c ts to d i f f e r e n t tre a tm e n t c o n d itio n s ,
and t h a t a d o u b le -b lin d procedure was not employed th ro u g h Â
out th e experim ent. A study i s needed to determ ine th e
e f f e c t s of Ribaminol on v e rb a l le a r n in g which p r e s e n ts th e
r a t i o n a l e f o r th e p re se n t study in v o lv in g human s u b je c ts .
Conclusions and R a tio n a le
What s ig n if ic a n c e and c o n c lu sio n s can we a t t r i b u t e
to th e s e r e s u l t s ? The p re se n t review has not found any
d i r e c t evidence im p lic a tin g RNA as th e memory engram. The
evidence s t i l l remains c ir c u m s ta n tia l. Commenting on t h e i r
own fin d in g s and th a t o f o th e r r e s e a r c h e r s , Glassman and
Wilson s ta t e d :
44
There I s s t i l l not enough In fo rm atio n to reach an
unequivocal co n c lu sio n concerning our r e s u l t s . The
t r a i n i n g ex p erie n c e o b v io u sly causes both a change
I n behavior ( le a rn in g ) and a chemical change i n th e
b r a in , but th e r e a re many p i t f a l l s in tr y in g to c o r Â
r e l a t e chem ical changes i n th e nervous system w ith a
b e h a v io ra l t r i g g e r . In p a r t i c u l a r , th e r e a re no d a ta
th a t e s t a b l i s h w hether e i t h e r of th e se changes i s th e
cause o f th e o th e r , o r w hether th ey might be u n r e la te d
resp o n ses to two d i f f e r e n t s tim u li. This u n c e r ta in ty
a f f e c t s a l l r e s e a r c h where an ex p erien ce e x e rts one or
more b e h a v io ra l, b i o l o g i c a l and chem ical re sp o n se s.
Thus i t i s not known w hether th e chemical response
has anything to do w ith th e le a rn in g p ro cess p e r se,
o r w ith a p ro cess i n c i d e n t a l to i t . I t i s t h e r e f o r e
extrem ely d i f f i c u l t to c o r r e l a t e our r e s u l t s w ith th e
c o n s o lid a tio n of memory, a lth o u g h i t i s extrem ely
tem pting to do so. The RNA might code f o r p r o te in s
t h a t may be in v o lv ed in th e c o n s o lid a tio n of long
term memory, p o s s ib ly by ren d e rin g permanent sy n a p tic
a s s o c ia tio n s th a t developed during s h o rt term l e a r n Â
in g . The p r o te in may be r e l a t e d to th e p e p tid e ( s )
p o s tu la te d to be in v o lv e d in th e m aintenance and r e Â
t r i e v a l o f memory [ 1970, p. 293].
The i n i t i a l purpose o f th e review of th e l i t e r a t u r e
was to determ ine i n f a c t t h a t RNA i s th e m olecular t r a c e of
memory. A fte r c a r e f u l review of th e l i t e r a t u r e , t h i s g o al
s t i l l remains d i s t a n t , alth o u g h t h e r e are some i n d ic a tio n s
t h a t p r o te in s y n th e s iz in g systems a re in v o lv ed . C a re fu l
a n a ly s is of th e many r e s e a r c h s tu d ie s c i t e d i n t h i s r e Â
view in d i c a t e s th e fo llo w in g :
1. The cause of t h e in c r e a s e in c o r t i c a l RNA appears
to be a s s o c ia te d w ith in s tru m e n ta l c o n d itio n in g .
2. S tr e s s appears to be one of th e f a c t o r s causing
t h i s in c r e a s e .
3. I t i s p o s s ib le t h a t le a rn in g may r e p re s e n t th e
c a u s a tiv e f a c t o r , but t h i s does not mean t h a t
le a r n in g r e q u ir e s RNA s y n th e s is , e s p e c i a l l y s in c e
le a rn in g appears to occur i n th e absence of RNA
s y n th e s is (Cohen & Barondes, 1966; Agranoff et
a l . , 1967, 1970).
4. There i s agreement from s e v e r a l s tu d ie s i n d i c a t Â
in g t h a t p r o te in s y n th e s is i s n e cessary f o r s h o r t Â
term and lo ng-term memory c o n s o lid a tio n (Barondes
e t a l . , 1966, 1970; A granoff, 1967, 1970).
5. The evidence from s e v e ra l s tu d ie s in d i c a t e s th e
d i r e c t involvem ent of RNA in sy n ap tic fu n c tio n s
(Kahn et a l . , 1970; Appel et a l . , 1970).
6. No d i r e c t evidence of th e involvement o f RNA i n
le a rn in g c o n s o lid a tio n has been found, a t l e a s t
th e c r i t e r i a I I and I I I o f Digman and Sporn
( 1967) have not been e m p iric a lly v a lid a te d .
The p o s it i v e r e s u l t s of s tu d ie s In v o lv in g human
s u b je c ts s u ff e re d from g ro ss lack of ex p erim en tal c o n tr o l
which i n v a l i d a t e s th e se r e s u l t s . These m ethodological
weaknesses range from th e " o n e - g r o u p - p r e te s t- p o s tte s t"
ty p e , to th e incom plete p r e s e n ta tio n o f d a ta ; from paÂ
t i e n t s ' own assessm ent f o r determ ining t h e i r "improvement,"
to th a t o f t h e i r r e l a t i v e s ' assessm ent; from t h e e x p e r iÂ
m e n te r's knowledge of which s u b je c t was under what t r e a t Â
ment, to t h e lack of a placebo c o n tr o l group. Thus, th e
is s u e of th e in g e s tio n of RNA to in c r e a s e le a r n in g and r e Â
t e n t i o n , i s s t i l l in c o n c lu s iv e .
The p re se n t experiment was an attem pt to i n v e s t i Â
g a te t h i s q u e stio n w ith a much more e f f i c i e n t and pow erful
d esig n in th e quest fo r an answer to t h i s q u e s tio n .
CHAPTER I I I
DESIGN AND METHOD
V a ria b le s
The Independent v a r i a b l e s a r e : th e drug Ribaminol
and a placebo of s im i l a r appearance I n capsule form.
The dependent v a r ia b le s a r e : le a rn in g , r e c a l l ,
r e c o g n itio n - c o r r e c t sc o re s to p a i r e d - a s s o c i a t e words.
S u b je c ts
G e r ia tr ic s u b je c ts , as determ ined by d a ta co n tain ed
i n t h e i r medical re c o rd s were s e le c t e d in t h i s stu d y f o r
s e v e ra l re a so n s:
1. They were c o n tin u o u sly h o s p i t a l i z e d and t h e r e Â
f o re s u b je c t to d i e t a r y c o n t r o l.
2. They were f u n c tio n in g c o n s id e ra b ly below e a r l i e r
le v e ls and th e r e f o r e t h e r e may be more "room" f o r
improvement to appear, as c o n tr a s te d to normal
s u b je c ts who may be fu n c tio n in g c o n sid e ra b ly
c l o s e r to th e c e i l i n g o f t h e i r a b i l i t i e s . T h is,
of course, i s on ly an assum ption, and i t may w ell
tu r n out t h a t s e v e re ly d e t e r i o r a t e d s u b je c ts are
not optim al fo r r e s e a r c h o f t h i s type.
47
48
T h i r t y - f i v e s u b je c ts were s e le c te d . T h e ir average
age was 62, w ith a range from 52 to 84. A com plete i n i t i a l
c l i n i c a l work-up was o b tain ed p r i o r to e x p e rim e n ta tio n and
fo llow ing i t . This in c lu d e d a n e u ro lo g ic a l exam ination
w ith EEG, CBC, u r i n a l y s i s , and EKG. The experim ent was
run i n fo u r r e p l i c a t i o n s of e ig h t s u b je c ts each; a p i l o t
study in d ic a te d t h a t e ig h t to te n s u b je c ts were about th e
maximum t h a t could be handled on any ex p erim en tal day. I t
took 2 days to com plete d a ta c o l l e c t i o n on any s u b je c t
under each tre a tm e n t.
P r e te s tin g of th e e n t i r e a c c e s s ib le p o p u la tio n of
about 260 s u b je c ts was done u sin g a memory t e s t c o n s is tin g
of d i g i t s . W echsler ( 1 9 5 5) re p o rte d c o r r e l a t i o n from .5 1
to .71 between d i g i t span and v ocabulary. Any i n d iv id u a l
who was ab le to respond to a c r i t e r i a ranging from 3 to 7
d i g i t s was in c lu d e d in th e a v a i l a b l e sample; and, from t h i s
a v a ila b le sample o f 104 t h a t met th e c r i t e r i a , 35 s u b je c ts
were randomly drawn. The r a t i o n a l e on t h i s sample was
based on th e s tr e n g th of s t a t i s t i c a l a s s o c i a t i o n t h a t th e
experim enter expected among th e d i f f e r e n t means. In t h i s
s p e c i f i c case th e experim enter expected th e experim ental
tre a tm e n t to account f o r a t l e a s t 25 p e rc e n t of th e v a r i Â
ance on th e c r i t e r i o n v a r i a b l e . The s tr e n g th o f a s s o c i a Â
ti o n and sample s iz e a re i n t e r r e l a t e d . Based on Hays'
( 1963* pp. 327- 3 2 9) form ula, th e sample s iz e n e c e ssa ry to
account f o r a t l e a s t 25 p ercen t of v a r i a t i o n in th e c r i t e r Â
49
io n v a r ia b le was a t l e a s t 27 s u b je c ts under each tre a tm e n t,
th u s th e r a t i o n a l e f o r a sample s iz e o f 35 s u b je c ts . Mean
comparison of Ss d iv id e d in groups A and B found no s i g n i f Â
ic a n t d i f f e r e n c e on the s e le c t io n c r i t e r i o n (see Table 4,
Appendix).
The Design
S u b jects were a ssig n e d to th e two tre a tm e n t c o n d iÂ
ti o n s in groups o f e ig h t by th e method of sy ste m a tic r a n Â
d o m iz a tio n . O ne-half o f th e s u b je c ts was a d m in iste re d
o r a l l y , under a n u r s e 's s u p e rv is io n , 250 mg c ap su les of
Ribaminol d a ily f o r 28 days p r i o r to e x p erim en tatio n . The
o th e r h a l f re c e iv e d a s i m i l a r s iz e placebo. A doubleÂ
b lin d procedure was used throughout th e experim ent.
M a te ria l
The stim u lu s m a te r ia l f o r th e experiment c o n s is te d
of two s e ts of p a i r e d - a s s o c i a t e s words. The words were
computer s e le c te d randomly from th e 5000 most f re q u e n tly
used words from Thorndike-Lorge (1944) l i s t i n g s . The P-A
words were p r in te d on a 3 X 5 index card i n bold l e t t e r s .
A sh eet o f paper w ith th e P-A l i s t i n g was used to keep
sco re of c o r r e c t responses f o r each t r i a l .
Procedure
A fte r being on t h e drug f o r 28 days, s u b je c ts were
brought i n d i v i d u a l l y to th e experim ental room f o r th e f i r s t
50
day of t e s t i n g . Ss were th en g iv en a l i s t of e ig h t p a ir e d -
a s s o c i a t e s , each p re se n te d on a 3 X 5 in d ex card and p ro Â
nounced by E. S _ a lso s a id the words alo u d on each p re s e n Â
t a t i o n . A fte r th e e n t i r e l i s t was com pleted, E p re se n te d
th e stim ulus word and asked S ^ to say th e response word fo r
each p a i r . Then a l l words were p re s e n te d again, follow ed
by a second t e s t . This sequence was re p e a te d f o r 10 t r i a l s .
The sequence o f th e terms was re o rd e re d a f t e r each t r i a l .
On th e second day, Ss were given only th e stim u lu s word of
each p a i r and th en t e s t e d fo r r e c a l l o f th e response term.
Only one t r i a l was given. Also, on th e second day, Ss were
p re se n te d w ith two terms on a c a rd , one o f which he had
p r e v io u s ly seen as the response term; th e o th e r was a new
term t h a t had not been p re v io u s ly p re se n te d to him. S _ was
asked to reco g n ize th e term he had seen b efo re i n th e exÂ
p e rim e n t. The response term was w r i tt e n on d i f f e r e n t p o s iÂ
ti o n s on th e c a r d s . Ss were a l e r t e d to t h i s s i t u a t i o n .
Only one t r i a l was given.
The c ro sso v e r procedure began t h e next day: RibamÂ
i n o l Ss were sw itched t o placebo and v ic e v e rsa . Twenty-
e ig h t days l a t e r an i d e n t i c a l procedure was follow ed w ith
a l i s t o f d i f f e r e n t words. The fo llo w in g day Sjs were given
a r e c a l l and r e c o g n itio n t e s t . D o u b le-b lin d procedures
were used throughout th e experim ent. The d i f f e r e n t groups
o f s u b je c ts were sta g g e re d throughout t h e experiment w ith
a t l e a s t one-day i n t e r v a l between groups.
CHAPTER IV
RESULTS
Three c r i t e r i a were used as in d ic a to r s of im proveÂ
ment in th e p re se n t experim ent:
1. Learning of e ig h t p a i r s of P-A words f o r te n
t r i a l s f o r a p o s s ib le t o t a l of 80 responses;
2. R e c a ll, one s in g le t r i a l of the e ig h t P-A word
l i s t ; and
3. R eco g n itio n , one t r i a l of the e ig h t P-A l i s t .
A fte r th e c o n c lu sio n of th e experiment th e d o u b leÂ
b lin d procedure was d is c lo s e d and i t was d isco v ered t h a t
c o n d itio n A was th e Ribaminol tre atm e n t and c o n d itio n B
was th e Placebo t r e a t e d group.
Data Analyses
Due to th e n a tu re of th e experim ental d e s ig n -
re p e a te d d e sig n , and th e te n o r o f the r e s e a r c h problem
asked, th e l o g i c a l s t a t i s t i c a l a n a ly s is c a lle d f o r was a
s i n g l e - f a c t o r ANOVA w ith re p e a te d measures on th e same e l e Â
ments (Winer, 1971). Thus th e s e le c t io n o f th e s t a t i s t i c a l
procedure throughout th e a n a ly s is of th e o b tain ed d a ta fo r
a l l th re e c r i t e r i a . S t a t i s t i c a l com putations were p e r Â
51
52
formed on a 3170 CDC computer a t th e C a l i f o r n i a S ta te UniÂ
v e r s i t y Computer C enter, N o rth rid g e , C a l i f o r n i a . The
o b tain ed mean sco res f o r th e le a rn in g ta s k were as fo llo w s:
Ribaminol Placebo
24.3143 23.6571
These sco res in d i c a t e th a t th e Ribaminol t r e a t e d group p e r Â
formed s l i g h t l y b e t t e r on the le a rn in g t a s k than th e P la c e Â
bo t r e a t e d group, but when s u b je c te d to th e ANOVA a n a ly s is
t h i s d if f e r e n c e was not s t a t i s t i c a l l y s i g n i f i c a n t . As i n Â
d ic a te d i n Table 1, th e d if f e r e n c e between th e two t r e a t Â
ments f a i l to su rp ass th e c r i t i c a l P 3 4 ) = ^« 1 3.
TABLE 1
ANALYSIS OF VARIANCE FOR THE LEARNING CRITERION
Source df M S F P
Between s u b je c ts 34 553.4555
W ithin s u b je c ts
35
78.4966
Drugs 1
7.5571 0.107 0 . 7 ^ 8
R esid u al 34 70.9395
T o tal
69
631*9521
Thus t h e d a ta f a i l to r e j e c t th e h y p o th e sis of no d i f f e r Â
ence i n le a rn in g between th e Ribaminol and Placebo t r e a t Â
ments .
53
On th e c r i t e r i o n of R e c a ll, th e mean s c o re s fo r th e
Ribaminol tre a tm e n t was 1.7714; th e Placebo tre a tm e n t was
1.9714. I t i s h e re noted t h a t th e Placebo t r e a t e d group
performed s l i g h t l y b e t t e r on r e c a l l than the Ribaminol
group. When th e s e groups were su b je c te d to t h e ANOVA
a n a ly s is no s i g n i f i c a n t d i f f e r e n c e was found as in d ic a te d
in Table 2.
TABLE 2
ANALYSIS OP VARIANCE: RECALL
Source d f M S F P
Between s u b je c ts 34
8.2748
W ithin s u b je c ts 35
2.4588
Drugs 1 0.7000 0.398 0.5392
R esid u al 34 1.7588
T o ta l 69 10.7336
The o b ta in e d F value did not exceed t h e c r i t i c a l
F 9 5 (1 3 4) = 4 .1 3 , i n d i c a t i n g th a t t h i s a p p a re n t d i f f e r Â
ence i n mean sc o re s was due to random f a c t o r s and not to
th e e f f e c t of th e ex p erim en tal tre a tm e n t of t h e Placebo
c o n d itio n . Again, th e d a ta on th e R e c a ll c r i t e r i o n f a i l e d
to support th e r e j e c t i o n of th e h y p o th e sis t h a t t h e r e i s
no d i f f e r e n c e on memory between s u b je c ts t r e a t e d w ith
Ribaminol o r a Placebo.
I
54
The means fo r th e R ecognition c r i t e r i o n , as ex Â
p e c te d , were g r e a t e r th a n t h a t of th e R e c a ll c r i t e r i o n .
The mean f o r th e Ribaminol tre a tm e n t was 5.4571> f o r th e
Placebo group, 5.5429. These o b tain ed v a lu e s c l e a r l y i n d i Â
c a te no b e h a v io ra l d if f e r e n c e s due to th e e f f e c t of th e
d ru g s. Table 3 summarizes th e ANOVA a n a ly s is of th e d a ta
f o r memory as in d ic a te d by th e R ecognition c r i t e r i o n .
TABLE 3
ANALYSIS OF VARIANCE: RECOGNITION
Source d f M S F P
Between s u b je c ts
W ithin s u b je c ts
Drugs
R esidual
34
35
1
34
6.9741
2.0807
0 .1 2 8 6
1.9521
0 .0 6 6 0.7946
T o ta l
69
9.0548
A n aly sis of th e d a ta found no s i g n i f i c a n t d i f f e r Â
ence between th e groups t r e a t e d w ith Ribaminol or Placebo.
The o b tain ed F value f a i l e d to su rp ass th e c r i t i c a l
F 3 4) = th u s in d ic a tin g t h a t th e h y p o th esis of
no d if f e r e n c e on R ecognition was not r e j e c t e d .
I
55
D iscu ssio n of R e su lts
The r e s u l t s o f t h i s experim ent I n d ic a te t h a t a
s y n th e tic RNA, Ribaminol, ta k e n o r a l l y by g e r i a t r i c subÂ
j e c t s , did not I n c re a s e v e rb a l le a r n in g , Improve r e c a l l o r
r e c o g n itio n sc o re s on p a i r e d - a s s o c i a t e s ta s k s . On a l l
th r e e c r i t e r i a , le a r n in g , r e c a l l and r e c o g n itio n , th e r e Is
no evidence In th e d a ta th a t would suggest th e enhancing
e f f e c t s o f RNA, under th e se c o n d itio n s , as an In d u cer of
th o se b r a in f a c t o r s c o r r e l a t e d w ith b e h a v io ra l I n d ic a to r s
o f le a r n in g and memory.
These r e s u l t s c o n tr a d ic t th e e a r l i e r s tu d ie s of
Cameron e t a l . (1958, 1961, 1963* 1966) In which th e y
found p o s i t i v e r e s u l t s w ith g e r i a t r i c RNA t r e a t e d groups.
As noted e a r l i e r (G aito , 1963)* th e se p o s it i v e r e s u l t s
might have been more r e le v a n t to n u t r i t i o n a l needs th a n to
le a rn in g o r memory. Also, m eth o d o lo g ical weaknesses such
as lack o f p ro p e r c o n tr o ls of confounding f a c t o r s , I n a p Â
p r o p r i a t e s t a t i s t i c a l a n a l y s is , q u e stio n th e I n t e r n a l vaÂ
l i d i t y of th e s e s t u d i e s . One of th e stro n g p o in ts o f the
d esig n used In th e p re se n t stu d y I s th e In c re a se d power
due to i t s i n t e r n a l v a l i d i t y .
The n e g a tiv e r e s u l t s of th e p re se n t study I s conÂ
g ru en t w ith th e Nodlne et a l . ( 1967), T a llan d e t a l . ( 1965)*
E ls d o rf e r e t a l . ( 1 9 6 8) and o th e r s ; In which g e r i a t r i c
brain-dam aged p a t i e n t s were t r e a t e d w ith r e l a t e d RNA subÂ
sta n c e s and no Improvement i n le a r n in g o r memory was found.
56
The r e s u l t s of th e p re se n t stu d y a lso c o n tr a d ic t
th e p o s it i v e r e s u l t s on memory of th e Gelfand e t a l .
( 1967) study In which th e y ad m in iste re d Magnesium pemoline
to 21 h e a lth y male s u b je c ts . These r e s e a r c h e r s a t t r i b u t e
th e p o s it i v e e f f e c t s o f t h e i r RNA t r e a t e d group to th e
a n t i - f a t i g u e e f f e c t s of th e drug. The ta s k o f th e p re se n t
study was not a fa tig u e -in d u c e d type because the q u e s tio n
concerned w ith was not o f f a tig u e but th a t o f an In d u cer
of v e rb a l le a rn in g and memory.
And f i n a l l y , th e r e s u l t s from t h i s experiment
c o n tr a d ic t th e Glaskey e t a l . ( 1968) study in which th e y
a d m in iste re d Ribaminol to r a t s and found i t was e f f e c t i v e
i n in c r e a s in g th e r a t e of a c q u i s it i o n and r e t e n t io n on an
avoidance ta s k . I t must be noted t h a t th e Glasky e t a l .
study s u ff e re d from g ro ss lack of adequate c o n tr o ls .
Another l o g i c a l e x p la n a tio n o f t h i s d if f e r e n c e i s t h a t
r e s u l t s from ex p erim en tal animal s tu d ie s , w hile su g g e stiv e ,
cannot be c o n sid e re d c o n c lu siv e evidence f o r humans. Most
anim al s tu d ie s have been done on g e n e t i c a l l y homogeneous
groups. In humans th e p r o b a b i l i t y f o r in d iv id u a l g e n e tic
in flu e n c e s on le a r n in g a b i l i t y remains a major u n reso lv ed
f a c t o r .
57
Summary
The p o s it i v e r e s u l t s of p rev io u s s tu d ie s in v o lv in g
human s u b je c ts s u ffe re d from g ro ss lack of ex p erim en tal
c o n tr o l which I n v a l id a te s t h e i r p o s i t i v e r e s u l t s . I n the
p re s e n t r e s e a r c h a determ ined e f f o r t was made to develop
a w ell c o n tr o lle d d esign. Many q u e stio n s were ex p lo red ,
f o r example: What i s the e f f e c t of re c e iv in g one tre a tm e n t
b e fo re or a f t e r th e oth er? Mean comparison o f d i f f e r e n t
s u b je c ts b efo re and a f t e r th e c ro sso v e r was perform ed in
o rd e r to a s s e s s th e e f f e c t o f th e ex p erim en tal tre a tm e n ts
on th e c r i t e r i a . These s t a t i s t i c s found no^ s i g n i f i c a n t
d i f f e r e n c e s between th e Ribaminol o r th e placebo t r e a t e d
groups (see Tables 5 and 6 in th e Appendix). Thus, th e
pow erful d e sig n used in th e p re se n t study in c r e a s e s c o n f iÂ
dence i n th e n e g a tiv e r e s u l t s o b ta in e d .
CHAPTER V
SUMMARY, CONCLUSIONS, AND RECOMMENDATIONS
Summary
Problem
This r e s e a r c h was concerned w ith th e presumed r o l e
of r ib o n u c le ic a c id s (RNA) i n th e improvement of v e rb a l
le a rn in g and memory. The re s e a rc h , ex p lain ed i n g r e a t e r
d e t a i l i n C hapter I , su g g ests d iv e rse t h e o r e t i c a l models
ex p la in in g th e mechanism whereby sen so ry experiences o f
e x te r n a l and i n t e r n a l worlds o f the i n d iv id u a l a re t r a n s Â
formed by th e organism in to r e p r e s e n t a t i o n a l p ro c e sse s th a t
symbolize e x p e r ie n ta l events du rin g le a rn in g .
Some r e s e a r c h e r s claim ed t h a t macromolecular s t r u c Â
t u r e s , s p e c i f i c a l l y n u c le ic a c id s (RNA), are th e source
f o r c a rry in g in fo rm a tio n and memory s to r a g e . Evidence from
human and anim al s tu d ie s su p p o rtin g and c o n tr a d ic tin g t h i s
fo rm u la tio n was p re se n te d i n d e t a i l i n Chapter I I . Due to
th e c o n f l i c t i n g r e s u l t s in e x p e rim e n ta l human and anim al
s tu d i e s , th e p re se n t stu d y i n v e s t i g a t e d the r o l e of RNA,
in a s y n th e tic form, i n th e improvement of v e r b a l le a rn in g
and memory.
58
59
Procedure
Following a p i l o t stu d y , 35 male g e r i a t r i c subÂ
j e c t s , age range 50 t o 80, were o r a l l y giv en 250 mg of RNA
(Ribaminol) o r a placebo fo r 28 days. These were then
a sse s se d fo r le a rn in g improvement usin g a l i s t of e ig h t
p a ir e d - a s s o c ia te s of most f re q u e n tly used words from
Thorndike-Lorge (19^4). Each p a i r of words was p re se n te d
v i s u a l l y on a 3 X 5 index c a rd . Each was giv en 10 t r i a l s .
Word sequence was re o rd e re d a f t e r each t r i a l . The fo llo w Â
ing day Ss were p re se n te d only w ith th e stim u lu s word and
t e s t e d fo r r e c a l l of th e response term; one t r i a l was given.
Also on th e second day, Ss were p re se n te d w ith two terms
on a c ard , one of which was a p r e v io u s ly seen response
term and th e o th e r a new term, and Ss were th e n asked to
reco g n ize th e term p re v io u s ly seen in th e experim ent; a
s in g le ♦ r i a l was g iv en . C o rrect sco res were recorded f o r
a l l th r e e c r i t e r i a . C rossover procedures began on th e
next day; RNA s u b je c ts sw itched to th e p lacebo and v ic e
v e rs a . Tw enty-eight days l a t e r an i d e n t i c a l procedure was
undertaken w ith a new l i s t of p a i r e d - a s s o c i a t e s . DoubleÂ
b lin d procedures were used th ro u g h o u t.
Hypotheses and R e su lts
Concerned w ith th e p rev io u s q u e stio n , s e v e ra l h y Â
po th ese s were fo rm u la te d :
Ho^ That RNA, Ribaminol, ta k e n o r a l l y , does not
in c r e a s e v e r b a l le a rn in g sco res on a p a ir e d -
a s s o c i a t e s t a s k .
The r e s u l t s f a i l e d to r e j e c t t h e n u ll h y p o th e s is . An ANOVA
t e s t f a i l e d to fin d s i g n i f i c a n t d iff e r e n c e a t th e .05 l e v e l
among s u b je c ts given e i t h e r an RNA or a p lacebo tre a tm e n t
as in d ic a te d i n Table 1.
HOg That PINA, Ribaminol, ta k e n o r a l l y , does not
i n c r e a s e R e c a ll (memory) sco res on a p a ire d -
a s s o c i a t e s t a s k .
The r e s u l t s as evidenced by an ANOVA t e s t , found no s i g n i f Â
ic a n t d i f f e r e n c e among RNA- and p la c e b o - tre a te d groups as
r e p o rte d i n Table 2.
Ho^ That RNA, Ribam inol, ta k e n o r a l l y , does not
in c r e a s e R eco g n itio n (memory) sco res on a
p a i r e d - a s s o c i a t e s ta s k .
The o b ta in e d r e s u l t s and the ANOVA t e s t f o r re p e a te d groups
found no s i g n i f i c a n t d if f e r e n c e s among th e RNA- and placebo-
t r e a t e d groups as s t a t e d in T ab le 3.
Thus, th e r e s u l t s f a i l e d to p rovide evidence fo r
th e enhancing e f f e c t s of RNA, Ribaminol, on v e rb a l le a rn in g
and memory o f g e r i a t r i c s u b je c ts .
Conclusions
The s p e c ific q u e s tio n asked a t th e onset o f the
p re se n t r e s e a rc h was: Does RNA (R ibam inol), ta k e n o r a l l y ,
Improve v e r b a l le a rn in g and memory o f th e aged? The d a ta
p ro v id e no evidence of th e enhancing e f f e c t s of t h i s speÂ
c i f i c RNA su b stan ce. Due t o the r e s u l t s o f th e experiment
I t must be concluded t h a t , under t h e s e c o n d itio n s , w ith
g e r i a t r i c p a t i e n t s , RNA (Ribaminol) does not enhance v e rb a l
le a rn in g o r memory.
The lo g ic a l q u e s tio n at t h i s p o in t i s : What are
th e se n e g a tiv e r e s u l t s implying? The re s e a r c h e r o f f e r s
th e fo llo w in g i n t e r p r e t a t i o n of th e o b ta in e d r e s u l t s :
1. I t could be t h a t t h e drug u sed , Ribaminol, I s
not e f f e c t i v e w ith severe m em o ry -d eterio rated
s u b je c ts . E m p irical evidence p re se n te d e a r l i e r
s tr o n g ly su g g ests such an i n t e r p r e t a t i o n . The
n e g a tiv e r e s u l t s o f RNA r e l a t e d su b stan ces such
as Magnesium pem oline, C y le r t, T ria p , e t c . ,
g iv e credence to such an i n t e r p r e t a t i o n . I t
co u ld be t h a t th e enhancing e f f e c t s of Ribaminol
I s more a p p r o p ria te to th e l e s s m em ory-deteriÂ
o ra te d s u b je c t.
One u n reso lv ed q u e stio n i n most b e h a v io ra l s t u Â
d ie s i s t h a t of comparing perform ance ta s k of
anim als w ith t h a t o f humans. In most animal
s tu d i e s , th e ta s k s are of th e m an ip u lato ry or
a c t i v i t y ty p e , which would suggest low er- and
m id -b rain fu n c tio n s in v o lv ed . Thus, any i n Â
g e ste d su b stan ce t h a t would in c r e a s e a t t e n t i o n ,
reduce f a tig u e o r s tim u la te th e organism to
perform , could cause an improvement i n perform Â
ance s c o re s . I t could be t h a t p o s i t i v e r e s u l t s
of RNA t r e a t e d anim als are a t t r i b u t a b l e , not to
th e in h e re n t le a rn in g or memory p o t e n t i a l of
the s t e r o i d , but to r e l a t e d m o tiv a tio n a l e f f e c t s
on th e organism . When d e a lin g w ith humans and
ta s k s in v o lv in g language, a b s t r a c t i o n , v e rb a l
le a rn in g and memory, we a re d e a lin g w ith beÂ
h a v io r c o r r e l a t e d w ith h ig h e r c o r t i c a l fu n c tio n s ,
and, as such, th e in g e s tio n of any su b stan ce w ith
in h e re n t m o tiv a tio n a l e f f e c t s on th e organism
might prove t o be i n e f f e c t i v e i n improving v e r Â
b a l le a r n in g o r memory. Thus th e n e g a tiv e
r e s u l t s of th e p re se n t stu d y . E m p irical support
fo r t h i s i n t e r p r e t a t i o n was d isc u s s e d p r e v io u s ly
(G elfand et a l . , 1967; T a lla n d & McGuire, 1 9 6 7).
A broader i n t e r p r e t a t i o n of th e p re se n t re se a rc h
i s i n terms of th e assum ptions on which t h i s exÂ
perim ent was co n ceiv ed . R e su lts of p rev io u s
s tu d ie s i n d i c a t e t h a t th e r o le o f RNA, so f a r ,
i s :
a. To r e g u la te th e p ro d u ctio n o f s t r u c t u r a l
p r o te in s . E x ten siv e p h y s io lo g ic a l s tu d ie s
over a long p e rio d of time le d N irenberg
( 1 9 6 5) to a r r i v e a t such a co n c lu sio n .
More r e c e n t l y , Kahan et a l . ( 1970) p re se n te d
evidence s u g g e stin g the r e s t o r a t i v e fu n c tio n
of such p r o te in s .
b. To m ediate th e p ro d u c tio n and r e g u la tio n of
enzymes (N iren b erg , 1965; Rich, 1 9 6 5).
S e ro ta et a l . (1972) s p e c i f i c a l l y p o in t to
n o re p in e p h rin e which i s in c re a se d w ith the
presen ce o f RNA.
c. To f a c i l i t a t e sy n a p tic tra n s m is s io n s (Appel
et a l . 1970; A granoff, 1970). Thus th e
r e s u l t s of th e p re s e n t r e s e a r c h would i n d i Â
c a te t h a t RNA's in flu e n c e on th e CNS, the
neuron, axon, d e n d r ite s , and synapse i s of
a s u p p o rtiv e , r e p le n is h in g , f a c i l i t a t i v e
n a tu re and not t h a t of a memory m olecule.
Recommendations
Most of th e re se a rc h done to determ ine th e e f f e c t s
of r ib o n u c le ic a c id s (RNA) on th e organism has been conÂ
cerned w ith I t s e f f e c t s on perform ance. F u rth e r re se a rc h
Is needed I n a t l e a s t two broad a re a s , p h y s io lo g ic a l and
b e h a v io ra l. The i n a b i l i t y to I n t e r p r e t o b tain ed r e s u l t s
In p rev io u s s tu d ie s s tro n g ly su g g ests t h i s c a te g o r iz a tio n
f o r f u tu r e r e s e a rc h .
P h y s io lo g ic a l S tu d ie s
1. To determ ine th e e f f e c t s of RNA on s p e c if ic
b r a in a re a s and t h e i r f u n c tio n s ;
2. To I n i t i a t e s tu d ie s o f d i f f e r e n t d u ra tio n fo r
e s t a b l i s h i n g the e f f e c t s of RNA I n b ra in a re a s
and f u n c tio n s ;
3. To a s c e r t a i n RNA's r o le In s y n a p tic tra n s m is Â
s io n , p r e - and p o s t-s y n a p tlc in flu e n c e s ;
4. To d esig n and c a rry out w ell c o n t r o ll e d
s tu d ie s f o r e s ta b li s h i n g th e p ro p e r dosage of
RNA fo r a given organism; and
5. To u n d ertak e p r e n a ta l s tu d ie s t o a s se s s th e
e f f e c t s o f RNA on m y e lin a tio n , d e n d r i ti c p ro Â
l i f e r a t i o n , and o th e r CNS development, w ith
p o s tn a t a l c o g n itiv e c o r r e l a t e s .
B ehavioral S tu d ie s
1. To pursue s tu d ie s using Ribaminol w ith d i f f e r
ent age groups o f In d iv id u a ls In o rd er to
a s se ss I t s e f f e c t s on v e r b a l and p e rc e p tu a l
motor le a rn in g t a s k s ;
2. To develop le a r n in g s tu d ie s t h a t d i f f e r e n t i Â
a t e between v e r b a l and p e r c e p tu a l m otor-type
ta s k s and the I n flu e n c e o f RNA on th e se ;
3. To conduct s in g le su b je c t s tu d ie s In o rd e r
to observe the c l i n i c a l and b e h a v io ra l e f f e c t
o f RNA o v er d i f f e r e n t le n g th s of tim e;
4. To un d ertak e s tu d i e s w ith o rganic b r a in Â
damaged I n fa n ts to determ ine RNA*s e f f e c t
on c o g n itiv e and psychomotor development; and
5. To I n i t i a t e s tu d i e s w ith m alnourished I n f a n t s
to a s s e s s RNA's e f f e c t on b ra in fu n c tio n s ,
c o g n itiv e , p h y s ic a l and psychomotor d ev elo p Â
ment .
R E F E R E N C E S
66
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30,000
1 9 4 4 7 "
A P P E N D I X
M E A N COMPARISON TABLES
75
TABLE 4
M E A N C O M PA RISO N FOR DIGIT SPAN SEIECTION TEST
Mean S.D. N
Group A
^.833 1.295
18
Group B 4 .2 9 4
1.0 Vf 17
t 1 .3 5 *
*Not s ig n if ic a n t a t .05 l e v e l .
TABLE 5
M E A N COM PARISO N O F G R O U PS BEFO R E CRO SSO VER
C orrect Responses
in Learning
Mean S.D .
C orrect R e c a ll
Mean S.D .
C orrect R ecogn ition
Mean S.D .
Drug 2 k . 333 2 2 .4 5 3 2 .7 8 2 .3 4 7 5 .5 0 2 .2 5 6
P lacebo 28.176 14.032 2 .1 7 6 2.270
6 .0 5 9 1 .9 8 3
t -.6 0 3 0
.1297 .7765
*Not s ig n if ic a n t a t .05 l e v e l .
TABLE 6
M E A N COM PARISO N OF G R O U PS AFTER CRO SSO VER
C orrect Responses
in Learning
Mean S.D.
C orrect R e c a ll
Mean S.D.
C orrect R ecogn ition
Mean S.D.
Drug 24.882 15.3 0 1 1 .6 4 7 2 .0 2 9
5 .4 7 1 2 .4 0 1
Placebo
19.389 17.783
1.7 7 8 2.415 5 .0 5 6 I . 83O
t
• 9770 -.1 7 2 9 < .5772
*Not s ig n if ic a n t a t .05 l e v e l .
76
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Britton, Augusto (author)
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The effects of an RNA polymerase in the improvement of verbal learning and memory
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