“W

LIB RA R Y
Michigan 503
University

  
 
     

  

This is to certify that the

thesis entitled
BEHAVIORAL AND PHYSIOLOGICAL EFFECTS

OF LATE-ONSET PROTEIN RESTRICTION
IN MALE HOLTZMAN RATS

presented by

Elizabeth Fabri Gordon

has been accepted towards fulfillment
of the requirements for

M. A. degree in Psychology

 

 

 

Date 4220/”: 414277

0-7639

BEHAVIORAL AND PHYSIOLOGICAL EFFECTS
OF LATE-ONSET PROTEIN RESTRICTION

IN MALE HOLTZMAN RATS

BY

Elizabeth Fabri Gordon

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF ARTS

Department of Psychology

1977

ABSTRACT
BEHAVIORAL AND PHYSIOLOGICAL EFFECTS

OF LATE-ONSET PROTEIN RESTRICTION
IN MALE HOLTZMAN RATS

BY
Elizabeth Fabri Gordon

Certain pOpulations of older peOple are affected by
late-onset dietary stress coupled with behavioral changes.
Using an animal model twenty, 200 day-old rats were placed
in pairs by similarity of body weight with one pair-member
receiving a low-protein (3 percent) diet and the other an
adequate-protein (24 percent) diet. Weights were monitored,
plasma protein levels determined and tests carried out for:
1) activity level, 2) exploratory behavior, 3) food pre-
ference, 4) motor-coordination and 5) learning.

Protein levels and weights were significantly greater
for controls as were results of motor-coordination testing.
Fifty percent of the low-protein rats became emaciated.
Exploratory behavior was significantly greater for the
adequately nourished animals. Low-protein rats made more
errors on oddity discrimination learning but differences
were not significant. Both groups significantly preferred
the adequate-protein diet when both were offered simul-
taneously. Results suggest that behavioral and physiologi-

cal changes in rats can result from protein-deficient diets.

ACKNOWLEDGMENTS

My sincere appreciation is expressed to Dr. M. Ray
Denny, my thesis committee advisor, for his continuous
academic support and encouragement throughout this study.

I would also like to thank the other committee members,

Dr. Raymond W. Frankmann, Dr. Abram M. Barch and Dr. Jenny
Bond, for their various contributions to completion of this
thesis.

Special thanks to Dr. Jenny Bond for her willingness
to learn about my discipline while teaching me needed
knowledge about hers, and to Ms. Ellen Rolig for her

technical assistance.

ii

TABLE OF CONTENTS

LIST OF TABLES . . . . . . . . .
LIST OF FIGUES . O O O O O O 0
INTRODUCTION . . . . . . . . . .

Physiological Findings . .
Behavioral Studies . . . .
Learning Studies . . . . .
Nutrition and Longevity . .
Nutrition of the Elderly .

METHOD 0 O C O O O O O O O O O 0
Subjects and Diets . . . .
Apparatus . . . . . . . . .
Procedure . . . . . . . . .

RESULTS 0 O O O O I O O O O C 0
Physiological Test Results
Behavioral Test Results . .
Learning Task Results . . .

DISCUSSION . . . . . . . . . . .

APPENDICES O O O O O O O O O O O
A. Test Data . . . . . . .
B. Description of Contents

During Study . . . . .
C. Information on Specific

BIBLIOGRAPHY . . . . . . . . . .

iii

of Diary Kept

Subjects . .

Table
l.

2.

LIST OF TABLES

Composition of Diets . . . . . . . . .

Performance of Male Holtzman Rats in an
Open-field O O O O O O O O O O O O O 0

iv

20

32

Figure

1.

LIST OF FIGURES

Activity wheel with digital counter.

attached cage can be closed off from wheel by

sliding to the left . . . . . . . .

Motor-powered rotarod with sandpaper-covered
drum (6" in diameter) and a catch net located
20 or

below. The drum can be rotated at 12,

30 rpm . . . . . . . . . . . . . . .

Oddity discrimination apparatus. Four

removable boxes, 2 white and 2 black,

can be inserted (not shown). . . . .

The

can be
inserted 3 at a time in various combinations.
Each box has a door of the same color that

Mean number of seconds by speed for the

experimental and control 83 on the rotarod,

motor-coordination apparaEus . . . . . . . . .

Mean number of grams per day for the

experimental and control rats when offered both

the 3 percent and 24 percent diets .

21

22

24

3O

33

INTRODUCTION

Numerous studies have addressed the problem of the
detrimental effects of dietary deficiency on various para-
meters. Certain investigators using animal models have
been particularly interested in behavior and learning
manifestations resulting from protein-calorie deficient
diets. The impetus for much of the early research on the
effect of protein-calorie malnourishment on physical growth,
learning and behavior was the identification of the health
problem, kwashiorkor, prevalent in Africa and other under-
deve10ped areas. Signs and symptoms of kwashiorkor include
irreversible stunting of physical growth, irritability and
retarded mental deve10pment (Levitsky and Barnes, 1973).
Factors influencing the early "critical period" of brain
deve10pment are believed to be the primary cause of this
latter problem and therefore studies have involved dietary

manipulation during the perinatal period.

Physiological Findings

 

Studies on the effects of protein deprivation on
immature laboratory animals have been carried out by many
investigators using a variety of techniques to effect
malnutrition and investigate its consequences. Richter

(1959) described a higher rate of amino acid incorporation

l

into the proteins of the developing newborn brain of the rat.
Laboratory investigations by Chow (1964) revealed that
dietary restriction of rat dams during pregnancy and lacta-
tion or during lactation alone resulted in significant
reduction in body growth in pups. This was permanent for
rats malnourished during the combined in utgrg and suckling
period and was the same for male and female pups. This
study was a relatively early one in this area of research
and approached the methods of feeding not from administra-
tion of carefully prepared laboratory diets of differing,
but well characterized, protein-calorie concentrations but
instead by use of a commercial laboratory feed which was
restricted in the amount given to some animals. This is a
major criticism when studies require diet manipulation.
Ingredients of commercially prepared diets, when analyzed,
have been found to be inconsistent with reported content
information (Greenfield and Briggs, 1971).

Mouse models were used by Randt and Derby (1973) who
found that severe nutritional restriction of the dams during
gestation and suckling resulted in mice with mean brain
weights that were 20 percent lower at birth than for pups
of dams fed normal diets. This developmental defect was
noted to be persistent when it was found that the weights
of the cerebellum of malnourished rats had a mean of only
86 percent of those of the control when studied at 600 days

of age.

Goodrick (1974) fed male mice diets containing 4
percent protein (casein) or 24 percent protein (casein) at
five weeks of age and varied the experimental conditions
by housing one, two, or five mice per cage. For all groups,
the body weights of mice fed normal-protein diets were
greater for group-caged than for isolated mice and body
weights of mice fed low-protein diets were less for group-
caged than for isolated mice. At 15 and 16 weeks of age
those mice fed low-protein diets engaged in more exploratory
behavior and were less emotional than mice fed normal-
protein diets. These results point out the necessity of
keeping the possibility of social-behavioral factors in
mind when interpreting data from this type of study.

Sobotka et a1. (1974) fed lactating dams a 12 percent
casein diet while controls received 24 percent casein.

The malnourished pups born to the 12 percent dams demon-
strated signs of retarded physical deve10pment including
reduced body_growth and delayed eye opening. Rat pups had
reached only 44 percent of the body weight of the pups from
control litters at the time of weaning. Detailed bio-
chemical analyses of brains from these animals showed a
marked reduction in cholesterol, DNA and acetyl-

cholinesterase.

Behavioral Studies

 

Behavioral changes described in humans suffering from

kwashiorkor include apathy and irritability (Cowley and

Griesel, 1964). Several workers have also looked for
behavioral effects of protein deficiency in laboratory
animals. Cowley and Griesel (1964) studied the maternal
behavior of the protein deficient parent generation of
rats. No significant behavioral changes in the protein
deficient group were observed except in nest building
which was more active than with control rats and in time for
returning pups to the cage when placed on a retrieval board
where the mean time was longer for the low-protein dams.
Studies of the first filial generation (male animals)
revealed that the protein deficient rats hoarded fewer
pellets than the control group and were slower in emerging
from their home cages. Differences between protein de-
ficient and control rats were marked on Hall's Open field
test in that the mean ambulatory score for the low-protein
rats was lower and the mean number of fecal boluses was
‘greater when compared to controls.

Frankova and Barnes (1968) continued the same line of
investigation with emphasis on behavior. They reported
that striking behavioral differences developed in groups of
rats which had been restricted in protein both before and
after weaning. During the 12 days of the experiment,
increased excitability was noted in these animals. Be-
havioral abnormalities were manifested as inadequate,
stereotyped movements and an inability to delay or ex-
tinguish fixed conditioned reactions. Those animals with

deficient protein intake in the pre-weaning period did not

show similar behavior patterns seen in the rats restricted
both before and after weaning.

Frankova (1973) studied the various social inter-
actions of malnourished young male rats in an observation
cage. The malnourished group had a delayed development of
social behavior. On the 49th day, control rats showed
increased exploration of the social stimulus, e.g., a
second rat was placed in the cage, whereas the malnourished
rats were markedly inhibited. It was suggested that the
disturbances seen in early social activities were caused
by the impaired development of neurosensory structures
secondary to the malnutrition and that, not only is there
a delay in behavioral development, there is a massive dis-
turbance of the social characteristics of the individual.
The protein-calorie deficient rat tries to avoid the new
social exposures that the normal animal seeks. Decreased
investigative behavior has also been noted in monkeys
(Strobel and Zimmerman, 1971). In a free operant chain
manipulation situation, protein deficient monkeys showed a
marked decrease in responding when novel objects were
attached to the chains, whereas performance increased in
the adequately nourished control group.

Pettus et al. (1974) as part of the extensive primate
research program at the University of Montana, investigated
the behavior of protein-malnourished monkeys and concluded
that the behavioral syndrome associated with protein-

calorie malnutrition may be improved by six months of

nutritional rehabilitation. Peregoy et al. (1972) found
that previously protein-malnourished rhesus monkeys showed
a marked preference for high protein food when compared to
adequately nourished controls.

Barnes et a1. (1968) studied postnatal nutritional
deprivations in rats to determine the effects on adult
behavior toward food and found total food intake was signi-
ficantly greater in the malnourished group compared to the
controls. When food was available for only one hour each
day, male rats fed the low protein diet had a three fold
increase in food spillage. There were no significant
differences in either food consumption or food spillage in

female rats when compared to controls.

Learning Studies

 

Barnes et al. (1966) deprived rats of food during the
first three weeks of life by increasing the number of rat
pups nursing one dam. Control litters contained eight
pups each. These animals were weaned at three weeks of
age and fed a low—protein diet (4 percent casein in one
group and 3 percent in the other) for eight weeks. The
four groups used were: 1) control, 2) nutritional depri-
vation by increasing the number of pups nursing, 3) nutri-
tional deprivation by feeding a low protein diet after
weaning, and 4) a combination group in which both pro-
cedures were used to.produce malnutrition. Males deprived

prior to and after weaning made significantly more errors

7

in a Y-water maze test of visual discrimination than did
females. This is the only study of which we are aware
reporting a sex difference on a learning measure. These
authors found that nutritional deprivation in early life
can cause a long lasting, possibly permanent retardation in
the development of learning behavior. They concluded that
motivational or emotional behavioral differences noted
among the treatment groups made it difficult to know the
relative contribution of drive versus capacity in the later
learning behavior.

Rats maintained on a low-protein diet from the time
of weaning made fewer errors than controls in a multiple-Y
maze when the reinforcement was protein-adequate food
(Griffiths and Senter, 1954). This study is of special
importance since it demonstrated that when food is used
as an incentive for learning in nutritional deprivation
studies, the malnourished animals may be more motivated to
perform. This consideration has been overlooked in pre-
viously reported studies where food was used as the rein-
forcement (Cowley and Griesel, 1959 and 1963).

Several studies using the protein-deficiency model
have resulted in different findings concerning the time of
greatest vulnerability to dietary stress in later adult
performance on learning tasks. Giurintano (1974), using
Wistar rats, found that postnatal protein deprivation
resulted in deficiencies in learning a difficult water

maze. No such deficits were found when dams of another

group of subjects were protein-deprived during gestation.
No learning deficiencies were found in a study by Frankova
and Barnes (1968) in avoidance conditioning learning, when
subjects were both pre- and postnatally malnourished.

Bush and Leathwood (1975) using Swiss white mice found
poorer performance in avoidance learning tasks for both

the pre- and postnatally malnourished groups, when compared
to controls. The species used, and the type and complexity
of the learning task may be important considerations when
findings in the literature fail to agree.

Levitsky and Barnes (1973) have delineated the strik—
ing similarities between the effects of early malnourish-
ment and environmental stimulus deprivation and have
suggested that the underlying mechanisms causing these phy-
siological and behavioral changes may be similar. These
investigators found that when both diet and environment
were manipulated, negative behavioral effects increased,
suggesting an interaction between these two variables
(Levitsky and Barnes, 1972).

Review of the above literature indicates that pre-
and postnatal dietary stress has detrimental physiological
and behavioral consequences in laboratory animals and these
effects appear to be analagous to those present in children
afflicted with kwashiorkor. Conclusions drawn from many
of the early studies are questionable since serious method-
ological problems, as mentioned above, make the findings

difficult to interpret. Consideration of the overall body

of literature in this area, however, indicates that peri-
natal malnutrition does adversely affect the organism,
even though these effects are inconsistent among studies

and are often poorly defined.

Nutrition and Longevity

 

The effects of diet on longevity have been studied in
rats by several investigators using protein and/or calorie
restriction models. McCay (1939) found that young rats
fed diets, restricted in calories only, experienced re-
tarded growth and increased life span. The rats were
restricted for 300, 500, 700 or 1,000 days before growth
to maturity was allowed. Rats from each of the four groups
were living when the last of the controls had died. McCay
suggested that this method could be used to produce very
old animlas for aging studies.

A study was designed to determine if intermittent
fasting in rats would cause longer life when compared to
controls fed ad libitum (Carlson and Hoelzel, 1946). The
three experimental groups were fasted as follows: one
day in four, one day in three or one day in two. The mean
life spans of all three groups exceeded that of the control
group and fasting one day in three was optimum. In
contrast to McCay's study little or no physical stunting
occurred using this intermittent fasting model. Carlson
and Hoelzel (1948) used an intermittent fasting design

combined with low residue or bulky diets and found that

10

bulk-formers added to diets fed ad libitum resulted in
increased life spans similar to those found when rats were
fasted one day in three. High fat diets of 22.7 percent fat
content caused a significant decrease in life span in

both male and female rats when compared to a group fed a
carbohydrate diet containing 3.4 percent fat (French et a1.
1953). The same rate of growth occurred in both experi-
mental groups even though fewer calories were consumed in
the high fat condition.

The effects of nutrition on longevity and onset of
disease were studied by Berg and Simms (1960). There were
three levels of food intake: 1) unrestricted, ad libitum,
2) 33 percent less than §d_libitum, and 3) 46 percent less
than ad libitum. Retarded growth was avoided by these less
severely restricted diets and only rats free from infection
were used so that the possibility of confounding nutrition
and infection variables would be minimal. Condition one
(ag_libitum) produced rats with large skeletal size and
obesity. No obesity occurred in the other two conditions
(33 and 46 percent), little skeletal retardation was noted,
and onset of disease was delayed as evidenced by post-
mortem autopsies and microscopic tissue examination. The
life span was extended (Berg and Simms, 1961) and the rats
living beyond 800 days were studied for the effects of
restricted diet on mortality rate, onset of disease,
appearance and behavior. The restricted group, when com-

pared to the unrestricted controls, weighed 40 percent less.

11

They were not, however, malnourished and when over 1,000
days old had the appearance of young, healthy rats. They
were more energetic than the obese §g_libitum animals who
developed weakness or paralysis of the hind legs and loss of
tail movement. The restricted condition continued the lower
mortality trend found in the previous study, and this was
attributed to the later onset of organ lesions.

Most research on the relationship between diet and
longevity has used lifelong dietary regimens. The underlying
assumption was that if the nutritional needs were not met
during the growing period, late-onset diet changes would
not influence life span. Ross (1972) studied the effects
of changing the types and amounts of food intake in
older male rats and found that the level of caloric restric-
tion and the protein-calorie ratio of various diets either
lengthened or shortened life expectancy. Longevity in-
creased in rats fed a limited diet with a protein-calorie
ratio of 1:5. This regimen of food intake caused a reduc-
tion in body weight. When other groups of animals were
fed diets that increased body weight, the length of life
was found to decrease. A program of life-long restricted
food intake had a greater effect on longevity, however,
even in the adult life of the rat the length of life was

significantly increased through dietary manipulation.

12

Nutrition of the Elderly

 

Many complex factors are involved in the nutrition of
the elderly human and in the relationship of diet to the
aging processes. Nutritional status of the elderly may be
influenced by prior cultural sets and decreased physical
mobility and vigor so that food behavior is adversely
affected. Malabsorption can be related to dental problems,
loss of gastric and enteric function, and diminished income
for food (Mann, 1973, Loeb and Howell, 1970a). Some aged
persons suffer from senile anorexia with complaints includ-
ing burning of the tongue, dry mouth, loss of taste and
difficulty in swallowing. When frequently associated
diseases presenting with these symptoms, such as carcinoma,
uremia and chronic infections, are ruled out the remaining
majority of cases are left undiagnosed as to cause. Vinther-
Paulsen (1952) evaluated the food intake of elderly
patients (67 to 96 years) and concluded that senile anorexia
may be due in some cases to malnourishment. Many patients
had a history of insufficient nutrition prior to the onset
of symptoms.

Loeb and Howell (1970b) summarized the relationship
between deficient diet and psychological performance. The
symptoms of subclinical forms of specific and general
dietary deficiency are: 1) loss of appetite, 2) fatigue,

3) irritability, 4) anxiety, 5) recent memory loss,
6) insomnia, 7) distractability, and 8) mild delusional

states. They pointed out that many of the symptoms have

13

been reproduced in human experimental research where adult
men were deprived of B-complex vitamins for 60-90 days.
This was found to result in confusion and lowered judgment.
The same picture can be seen with older peOple on poor
diets or with psychiatric syndromes such as in schizo-
phrenics who eat poorly. Furthermore, these researchers
(Loeb and Howell, 1970c) pointed out that, in addition to
disease, the ingestion of drugs (prescription and non-pres-
cription), alcoholism and depression may influence nutri-
tion in the elderly.

Clinical observations of nutritional deficiency in
the aged have rarely been supported with research data.
Corless (1973) reviewed the hospital admissions of elderly
persons in England. He stated that the precise nutritional
needs of old people are unknown but did report that malnu-
trition in one form or another is very common in hospitalized
individuals. An editorial in the British Medical Journal
(1974) stated that although severe malnutrition was uncommon
in the elderly, certain individuals were clearly at risk.
These included people with arthritis who are unable to shop
or cook or confused females who may be able to cook for
their husbands and yet will not eat themselves.

One of the largest nutritional studies in the elderly
was carried out in Russia with 27,181 individuals, 80 years
and older (Chebotaryov and Sachuk, 1964). The majority of
these individuals ate moderately and were found to be lean

on physical examination. Few other details are given except

14

that few vegetarians or consumers of large amounts of meat
were found in the group. Schlenker et a1. (1973) stated
that no good longitudinal study has been carried out, and
further noted that completely satisfactory criteria for
determining optimum nutritional status are not available.

Kelly et a1. (1957) studied females 40-80 years of
age, and upon reevaluation seven years later found mortality
to be greater when dietary intake contained 40 percent or
less than the recommended daily allowance (RDA) for one or
more nutrients.

Whanger and Busse (1975) working with psychiatric
disorders in the aged, reported that over 70 percent of
hospital admissions were eating doubtfully or grossly
inadequate diets. Fifty percent of these patients had
borderline to low plasma levels of folic acid, and 12 per-
cent had borderline to low levels of vitamin B12. They
noted that these two dietary deficiencies have been
associated with certain psychiatric disorders, and empha-
sized the problem of determining in these psycho-geriatric
patients, whether the deficiencies cause the illness or
result from it.

Previous studies on behavioral effects of malnourish-
ment have been primarily concerned with perinatal nutri-
tional dietary stress, following the discovery of the rela-
tionships of diet to kwashiorkor. Studies on diet and
longevity have mostly involved life-long dietary programs

and have not been specifically concerned with behavior.

15

The available information on human nutrition among the
elderly suggests the need for well controlled, systematic
studies to determine the relationship between diet and
behavior when the nutritional deprivation occurs in later
life.

Use of animal models allows for more careful control
of the relevant variables and is of particular value where
severely deficient diets are deliberately imposed upon an
organism so that the effects can be evaluated. The Holtzman
rat strain was selected for this study since its life ex-
pectancy is less than other strains. Semi-purified diets
were used so that diet composition could be monitored care-
fully throughout the study. The selection of tests was
made on the basis of anticipated changes in the animals,
resulting from the experimental condition. Each can be
related to the expected differences between the protein-
restricted group and the controls as follows: 1) activity
wheels to assess general activity, 2) the open field was
used to determine if exploratory behavior would be affected
by low-protein intake, 3) the rotarod was used to determine
if motor-coordination differed between the groups since it
was believed that the low protein group would experience
loss of muscle mass, 4) the food-preference test was used
to demonstrate the presence of sensory capacity in older
animals to distinguish between diets of different composi-
tions and to test for possible "specific hungers" in animals

on restricted diets, 5) the oddity discrimination task was

16

selected as the test used to find the presence of learning
and/or performance differences between the groups. This is
a complex task and would thus remove the possibility present
when using simpler learning tasks where all animals may
perform well, and possibly differences would not be detected
(Bachelder and Denny, 1976).

Initially, a protein model of dietary restriction was
used since protein foods are the most expensive in the diet
and older peOple may have more difficulty with consumption
of protein foods such as meats (e.g., presence of dental
problems) and milk products due to lactose intolerance.

The specific aims of this study included the following:

1. To establish an animal model that could be used

to evaluate behavioral effects of late-onset
dietary stress.

2. To evaluate behavior in aging rats using tests

for exploratory behavior (open-field), motor-
coordination (rotarod) and learning (oddity
discrimination).

3. To evaluate age-appropriateness of selected tests.

4. To define problems associated with using older

animals in nutrition-behavior studies.

The general hypothesis tested was that late-onset
dietary stress in rats can result in physiological,
behavioral and learning differences when compared to
adequately nourished controls. Past workers in the area of

the relationship between nutrition and behavior, have

17

generally stated that it is unknown in nutrition-behavioral

situations whether malnutrition is the cause of psycho-

social problems or results from them.

The results of testing the above hypothesis would

demonstrate that changes observed in older living organisms

can be attributed primarily to experimentally manipulated

diets.

The specific hypotheses tested were as follows:

1.

Plasma protein levels could be lowered in older
rats fed a low protein diet.

weights of low-protein fed animals would be lower
than pair-members fed an adequate protein diet,
even though the diets were made isocaloric and
caloric intake for pair-members was approximately
equal.

Performance on a motor-coordination task would be
poorer in protein restricted animals compared to
controls fed adequate amounts of protein.
Activity levels would be greater for the well-
nourished controls than the experimental group.
Exploratory behavior would be greater in the
controls compared to the experimental group.

Rats fed a low-protein diet would show a pre-
ference for an adequate diet when given both to

choose between.

18

7. Adequately nourished rats would do better on a
complex learning task, when compared to rats

fed protein-restricted diets.

METHOD

Subjects and Diets

 

Twenty, 200 day-old male Holtzman rats were placed in
pairs by similarity of body weight. The weight range for
all §$ was between 495 and 568 grams, and between zero and
four grams for matched pairs. One of each pair was randomly
selected to serve as the experimental 3 percent, low-protein
§_and the other pair-member was the 24 percent adequately
nourished control (see Table l for composition of diets).
The SS were pair fed as described by Yang and Mickelsen
(1972), where the experimental rats were fed ad_1ibitum and
the controls fed the amount eaten by their pair-member the
previous day. Tap water was available ad_1ibitum for all

§3 and the laboratory was on a 12:12, light:dark cycle.

Apparatus

 

Testing equipment included: 1) standard activity
wheels, adapted for larger rats (Figure 1), 2) a square
open-field with 64, six inch squares, enclosed by a 12"
high plywood wall, 3) a rotarod motor-coordination apparatus,
consisting of a sandpapered drum (6" in diameter) mounted
on a rod attached to a motor that could be run at three
Speeds, 12, 20 or 30 rpm (Figure 2), and 4) an oddity-
discrimination apparatus with a start box, choice area and

19

 

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The

attached cage can be closed off from wheel

Activity wheel with digital counter.
by sliding to the left.

Figure l.

Dire<
of d:

Figu

22

Direction
of SS
Direction
of drum ——— i_- F/‘
I!

 

 

 

 

 

 

 

 

 

 

Figure 2. Motor-powered rotarod with sandpaper-covered
drum (6" in diameter) and a catch net located
below. The drum can be rotated at 12, 20 or
30 rpm.

23

end chamber. Four removable boxes, two black and two white,
were inserted three at a time in various combinations, e.g.,
WBB, BWB, WBW (Figure 3). All §s were housed in individual,

wire-bottomed cages (8x8x10).

Procedure

 

All §s were pair-fed and weighed weekly for 10 weeks.
Each S was then etherized and a tail blood specimen taken.
Plasma protein levels were determined using the procedure
described by Lowry et a1. (1951). Following the blood

analysis, testing was begun.

Test 1, General Activity: The Ss were approximately

 

300 days old at the beginning of this test and had been
under the pair-feeding condition for 100 days. Ten activity
wheels were available and, therefore, the 10 experimental
and nine control gs were randomly assigned to one of two
groups. The first group of gs was housed and fed in
activity wheels for eight days. Food and water were
available during the 12 hour light cycle and access to the
wheel only during a 12 hour dark cycle. The number of
revolutions for each S was recorded at the end of each 12
hour period. This procedure was followed for the second

group immediately after testing group one.

Test 2, Exploratory Behavior: The gs were approxi—

 

mately 320 days old at the time of testing, had been under

the pair-feeding condition for 120 days and were all tested

24

 

 

 

 

 

 

 

 

 

 

 

’2“
End -H
Box 5
A. m
g H
o
o m
B
~v u
m
m
n
m
(Plexiglas)
Choice
Area
0
'5
ill
l' A
- m
' 5 Start
c: BOX
m
m
u
0
.a m
U
0 U
o ' m
3 O
V J:
m

 

 

 

 

 

Figure 3. Oddity discrimination apparatus. Four removable
boxes, 2 white and 2 black, can be inserted 3 at
a time in various combinations. Each box has a
door of the same color that can be inserted (not

shown).

25

on the same day. There were nine experimental and seven
control §5 tested. Each gs was placed in the corner of
the opern-field, facing the vertical line and observed
for three minutes. The total number of squares and the

number of internal squares entered were recorded.

Test 3, Food Preference: The §S were approximately

 

327 days old and had been pair-fed for 127 days. Eight
experimental and seven control gs were tested. Two food
cups, one containing the 3 percent protein diet and the other
the 24 percent protein diet, were placed in each cage and
fastened to prevent movement. The cups were rotated from
side to side (to control for position bias) for four
successive days. The amount eaten from each cup was

recorded at the end of each 24 hour period.

Test 4, Motor-Coordination: The §s were approximately

 

350 days old and had been pair-fed for 150 days at the
time of testing. Eight experimental and seven controls
were tested and the procedure was completed in one day.
Each §_was tested on the rotarod drum as described by
Overmann (1976). There were three practice trials on the
drum rotating at 12 rpm, followed on the same day by three
test trials, one each at 12, 20 and 30 rpm. The number

of seconds that the § remained on the drum was recorded.

Test 5, Oddity-Discrimination Learning: The habitua-

 

tion to the apparatus was begun when the §s were 450 days

26

old and had been under the pair-feeding condition for 250
days. Following eight days of habituation, the test trial
procedure was started. Seven experimental and five controls
were run during a three week period. All gs were initially
habituated to the apparatus following six hours of water
deprivation. Each S was placed in the start box and
allowed to move about freely. Artificially sweetened water
was available in each of the three end boxes. This proce-
dure was repeated daily until §5 were drinking within three
minutes after being placed in the start box. The gs then
had 12 trials each day and all combinations (WBW, WBB,

WWB, BWB, BWW and BBW) were randomly presented twice.

The water reinforcement was available in the odd box only.
On the first four days, there were six randomly deter-
mined forced choices where only the odd-colored box was
open. Over the four days there was an equal number of
black and white forced correct choices. If the §_entered
an incorrect box during any of the test trials, the doors
of the two remaining boxes were lowered to prevent a double
error in any one trial. The §_was allowed 30 seconds to
leave the incorrect chamber, afterwhich he was picked up

at that point and that trial was ended. If the §_1eft the
incorrect chamber, that door was lowered and the §_picked
up after 20 seconds. When the S entered the correct box,
that door was lowered and drinking was allowed for five
seconds. A variable intertrial interval (ITI) was used

(30 seconds to one minute). The acquisition criterion was

27

10 correct responses in 12 trials. The rate of habituation
and the number of correct choices were recorded.

The §s were kept on the pair-feeding condition for
three weeks following Test 5. They were then weighed and
sacrificed. Their approximate age at that time was 500
days, and they had thus been under the experimental diet
condition from midlife to end of life expectancy. A bone
density analysis was performed on these and all gs that had

died and been preserved during the completion of the study.

RESULTS

The physiological measures, including plasma protein
levels and weights for intact pairs, were statistically
analyzed using the Student's t test for matched pairs.

The remaining test data were analyzed using the t test

or analysis of variance for independent groups. Although
the physiological data were analyzed using the matched
pairs t test, the matching was disregarded in the analysis
of the behavioral and learning data, since §$ from both
treatments were lost during this part of the study. This
permitted data from the remaining pair-member to be used,
thus keeping as many degrees of freedom for the analyses
as possible. The reason for using both methods of data
analysis was that it was unlikely that these psychological
tests would necessarily show differences between pair-
members to a greater degree than among members in each
treatment. weight was the only criterion for matching.
The subjects were placed in pairs so that the appropriate
pair-feeding procedure could be carried out to test for
physiological differences between pair-members. A minimum
level of significance of (p‘i .05) was set prior to the

study, and non-directional tabled values were used.

28

29

Physiological Test Results

The analysis of both plasma protein levels and weights
was performed on data collected after the study has been in
progress for 10 weeks and prior to the loss of any gs. The
plasma protein levels were significantly different (t = 2.85,
df 9, p‘: .05), for matched pairs and as expected the experi-
mental, low-protein §3 had lower levels. The weights
between pair-members were significantly different (t = 2.78,
df 9, p i.-°5)I and as predicted, the experimental pair-
member weighed less than his control.

An analysis of the data from the activity wheel
test, used for general activity, showed no significant
differences; however the adequate protein group did have a
higher group mean than the protein restricted gs.

A 2 x 3 (diet by speed) analysis of variance was used
to analyze the rotarod, motor-coordination data (Figure 4).
The results showed poorer performance for the experimental
group by diet (F = 28.37, df l, 13, p < .001) and speed
(F = 44.24, df 2, 26, p < .001) with a significant inter-
action between diet and speed (F = 3.79, df 2, 26, p i .05).

Fifty percent of the protein restricted rats became
emaciated in outward appearance during this investigation.
It was noted at the time of sacrifice that seven experi-
mental and three control gs survived. A Chi-Square test
showed that the differences between survival rates

approached significance (x2 = 3.2, df l, p i .10).

30

 

 

 

3.0-,
53
c 2. ..
o 0
o
o
m
u
g
5
c
a
m
m
2

1.0

: t l

12 20 30
Speed (rpm)
Figure 4. Mean number of seconds by speed for the experi-

mental and control §$ on the rotarod, motor-
coordination apparatus.

31

Behavioral Test Results

 

A one way analysis of variance was performed on the
open-field test data (Table 2). Exploratory behavior was
found to be significantly different between the experimental
and control groups (F = 5.01, df l, 14, P.i .05), with the
adequate-protein control gs entering a greater number of
squares than the low-protein, experimental S3.

The food-preference data were analyzed for each group
using Student t to test for a significant preference for the
adequate-protein diet in both_groups (Figure 5). This was
found to be significant for the experimental group (t = 7.102,
df 7, p §_.001) and the controls (t = 2.77, df 6, pg: .05).
Both groups, when given a choice between diets preferred the

24 percent, adequate-protein mixture.

Learning Task Results

 

Two gs from the experimental group would not run and
therefore were dropped for purposes of data anlysis. It was
noted that on day three of habituation the six controls
were drinking within eight minutes after being placed in the
apparatus (An eight minute time limit was preset as the
maximum habituation time per day), while only three of the
seven experimental §s were drinking within that time. The
differences in rate of habituation were not significant
however, in general there was less exploratory behavior in
the apparatus for the experimental group as compared to the

controls. Since exploratory-approach behavior in a novel

32

Table 2. Performance of Male Holtzman Rats in an Open-fielda

 

 

 

Diet N Numbers of Squares Crossed
Control 7 88.86 : 9.00b
Protein-Restricted 9 65.88 + 5.78c

 

aAnimals were 320 days of age at testing and had been
on respective diets for 120 days.

bMean :_standard error.

cSignificantly less than controls (p < .05).

Mean number of grams per day

Figure 5.

30.

N
O
r

10

 

33

\. 24% Protein Diet

Con.

 

  
 

3% Protein Diet

Con.

" ----

Days

Mean number of grams per day for the experimental
and control rats when offered both the 3 percent
and 24 percent diets.

flak—w“ —

 

34

situation, e.g., the oddity-discrimination apparatus, is

theorized to be part of an organism's innate behavior and
a necessary initial component to learning a task, a lack

of or diminished amount of such behavior is an important

consideration (Denny and Adelman, 1955).

The data recorded during testing for the five of the
seven experimental subjects that would run and the five
controls were analyzed using the Student's t test. No
significant differences were found between the groups for
the number of correct choices made, however, the controls
made fewer errors.

A strong preference for black end-box choice was
noted to be present in both groups during habituation.

A second t test was therefore performed to determine if
this initial preference persisted throughout the study.

It was found that the experimental group maintained a
significant black preference (t = 4.07, df 8, p :_.01)
throughout testing, compared to the controls. This is fur—
ther evidence that the experimental group had a tendency to

avoid novel situations.

DISCUSSION

The general hypothesis of this study, stating that
late-onset dietary manipulation can result in behavioral
and physiological changes, was supported by the findings.
Significant effects were found on the following measures:

1) plasma protein levels, 2) weights, 3) performance on the
rotarod, 4) exploratory behavior, 5) food preference and

6) the number of white end boxes entered during the oddity
discrimination learning task. Differences on the activity
level measure, and number of errors on the oddity learning
task were in the predicted direction but were not
significant.

Since this was a preliminary study in the area of
the relationship between nutrition and behavior in older
organisms when the dietary insult occurs in later life,
and is perhaps the first such study using an infra-human
model, these results cannot be compared with many previous
findings. A major concern in studies where behavioral pro-
blems and poor nourishment have been found to occur together,
has been whether nutritional deficiencies cause the problems
or result from them (Vinther-Paulsen, 1952; Loeb and Howell,
1970b, Schlenker et a1., 1973; Whanger and Busse, 1975).

The results of the present study suggest, that under certain

35

36

circumstances, behavioral changes can result from dietary
distress.

It was of great concern in this investigation to
establish a methodologically sound research design in this
area since poor methodology has been a major criticism
of many of the perinatal nutrition-behavioral studies in
the past (Greenfield and Briggs, 1971). Specifically,
diet composition was well controlled, and the diets were
made isocaloric. The pair-feeding procedure was used so p
that protein was the primary independent variable and
was not confounded with caloric intake. All necessary
nutrients were included in both diets, and drug therapy
was not used for actue illnesses, since drugs have been
found to interfere with metabolism of nutrient intake and
behavior.

The methodological oversights in setting up this
study included the failure to get baseline data for plasma
protein levels and the failure to assess levels at the
conclusion of the study. These data would have allowed a
longitudinal comparison of the plasma levels of the experi-
mental group, as well as the controls. Had levels become
lower in the experimental group over time, this would have
been good evidence that protein was the primary variable
resulting in behavioral differences between the low-protein
and adequate-protein groups.

If further research is to be continued in this area,

it is pertinent to review some of the practical aspects

37

of this investigation. The study demonstrated that older
male Holtzman rats can survive extended feeding of meal
diets and therefore can be kept on well controlled
nutrient concentrations. The sensory capacity for taste
and/or smell of these animals at approximately two thirds
of their life expectancy, was sufficient to distinguish
between the 3 percent low-protein diet and the 24 percent
adequate-protein diet. This information indicates that
food preference studies for older rats can be done. Con—
founding of age related variables with test results, such
as occur with the increased probability of disease in
older organisms, must be considered when determining the
number of subjects to be used. It is necessary to estab-
lish the criteria for eliminating subjects that become ill
during the study, prior to its start, e.g., those apparently
unrelated to the experimental conditions.

Tests that have been used previously to assess
behavior on various parameters have been used primarily
with younger laboratory animals than those in this study.
It is important, when testing naive older animals, that
age-appr0priateness of the tests be considered. If for
instance, a fear response was elicited on the rotarod
from these rats, this technique was not measuring motor
coordination as was intended. When this can be determined
or it is strongly suspected, an alternate test, such as the
observation of locomotor performance on a narrow board,

might be a more appropriate test for measuring motor

38

coordination. Methods for determining age-appropriateness
of tests are essential if specific behaviors are to be
evaluated and should be determined for all tests used.
Further studies in this area, based on these findings,
can be done using various nutrition deprivation models,
e.g., protein calorie malnutrition model (PCM). The consi-
deration of time needed to run an appr0priate battery of
tests may make it necessary to use a strain of rat with a
longer life span than the Holtzman. This is especially
true for learning tasks that may take hundreds of trials
before differences can be detected. It can be predicted
from previous nutritional research (Mills et al., 1969),
that many nutritional models, e.g., PCM, trace element
depletion, and various vitamin deficiencies can cause
faster physiological effects than the protein-restricted
model used here. If it can be demonstrated that behavior
is also affected, using one of these faster models, it
would be possible to design a study to include nutritive
rehabilitation and subsequent repeated tests to see if
reversal of effects would occur. It would, as evidenced
by findings from various studies such as Barnes and co-
workers (1966 and 1968), be advisable to do this type of
study using both male and female rats to determine the

presence or absence of sex differences.

APPENDICES

APPENDIX A

TEST DATA

APPENDIX A

TEST DATA

Plasma Protein Levels at 10 Weeks
Blood samples were collected, plasma was separated from cells

and analyzed. Eight of these analyses were repeated.

 

 

Subject Number Grams/100 ml.
1 10.70
2 13.90
3 7.11r
4 7.044
5 9.45
6 12.29
7 8.90
8 10.24
9 7.39r

10 7.60r
11 8.95
12 9.86
13 6.52r
14 6.79r
15 7.62
16 12.05
17 7.05
18 7.62
19 8.97r
20 9.16r

r indicates repeated analysis.

NOTE: Throughout appendices all odd numbered §$ refer to
those under the experimental condition, and all even num-
bered subjects were controls.

39

 

40

Weights for all S3 at 10 weeks from beginning of the experi-
ment and prior to the start of testing. These weights have

been corrected for initial differences between pair-members.

 

 

Subject Number Weight in Grams
1 405
2 410
3 462
4 436
5 501
6 506
7 501
8 502
9 471

10 514
11 487
12 547
13 344
14 416
15 528
16 552
17 490
18 496
19 452

20 528

Motor coordination

41

Experimental gs by speed (N=8)

R1

1.4

0.9

42

Open-Field
Experimental §s (N=9)

Number of Squares Entered

 

60
46
76
66
63
65
72
102

43

Control §S (N=7)

Number of Squares Entered

 

63
117
96
86
118
57

85

43

Food Preference
Experimental mean number of grams intake for each of four

days when offered both diets (N=8)

 

 

Day 3 Percent 24 Percent
1 6.250 26.625
2 0.375 21.250
3 1.000 20.375
4 0.875 21.000

TMumm . nm

Control mean number of grams intake for each of four days

when offered both diets (N=7)

 

 

Day 3 Percent 24 Percent
1 12.429 21.571
2 4.857 17.857
3 5.429 21.571

4 4.286 23.429

44

Oddity Discrimination
Number of correct responses for the experimental and control

rats (120 trials N=5)

 

Experimental Control
47 46
35 47
33 46 I
47 51
50 50 g
F

Number of white end boxes entered by experimental and

control rats (120 trials N=5)

 

Experimental Control
13 50
36 56
22 29
21 50
12 47

NOTE: For these analyses, two experimental animals were
dropped since they would not habituate to the testing
apparatus.

APPENDIX B

DESCRIPTION OF CONTENTS OF DIARY KEPT DURING STUDY

APPENDIX B

DESCRIPTION OF CONTENTS OF DIARY KEPT DURING STUDY

A diary of notations was kept daily from the beginning
of the experiments until the beginning of the last test
(oddity discrimination) at which time notes were made,
when necessary, on the raw data sheets. Recordings in-
cluded Efs impression of each rat's general physical condi-
tion and behavior. Specifically anorexia, fluid intake,
hair loss, appearance of stools, general activity, signs
of chronic respiratory distress (CRD), acute and chronic

illness and handling difficulties, were noted.

45

APPENDIX C

INFORMATION ON SPECIFIC SUBJECTS

APPENDIX C
INFORMATION ON SPECIFIC SUBJECTS

Three controls within the first week of the experi-
mental condition (pair-feeding) showed signs of acute-onset
illness including anorexia and loose stools. A notation
was made at that time that adjustment to the 24 percent
diet needed to be considered as the possible cause. Two of
these animals recovered, however number 8, a control rat,
remained intermittently ill throughout the investigation
until his death at 353 days of age. It was for this reason
that this subject was omitted from all data analyses.

The procedure used for animals that became ill during
the study was to isolate immediately for overt CRD
symptoms while keeping all other variables approximately

the same as under non-isolated conditions.

46

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