Animal model of pathological behavior

The present invention provides a phenotypically stable altered animal which exhibits unprovoked rage reaction indicative of high emotionality, stereotyped behavior and is useful as a model of psychotic behavior. The animals of this invention (F.sub.2) were obtained by inbreeding mature offspring (F.sub.1) of rats whose mothers received high tryptophan diets during their third trimester of gestation. There is also an increased body weight which evidences physical alteration. The present invention also relates to the preparation and use of animal models for the study of psychosis, pathological psychotic behavior in humans and the effects of tryptophan in successive generations. The prenatal effects of tryptophan on later generations can also be used to increase the body weight of experimental animals and livestock.

FIELD OF THE INVENTION 
The present invention relates generally to animal models of pathological 
behavior and, more particularly, to a phenotypically altered animal. 
Specifically, this invention relates to an animal, bred from the F.sub.1 
offspring of prenatally tryptophan-dieted rats, with behavioral 
characteristics resembling psychosis in humans. 
BACKGROUND OF INVENTION 
Psychosis is a general term used to describe mental illness which results 
in the severe disturbance of brain function, including emotion and 
intellectuality. The study of psychosis and its underlying causes and 
treatments are thus essential to society. 
Animals exhibit various behavioral characteristics which researchers have 
used to select animal models for study. As an example, emotional 
instability is associated with psychosis; thus behavior indicative of this 
characteristic has been used as a criterion for selecting appropriate 
animal models. For example, in his review of articles on emotional 
stability, Archer, J., Anim. Behav. 21:205-35 (1973) concluded that low 
emotionality in rats accelerated more exploration (rearing) and high 
emotion inhibited exploration. Additionally, Broadhurst, P. L., Behav. 
Genetics, 5(4):299-319 (1975) and Hall, C. S. J. Comp. Psychol. 18:385-403 
(1934) indicated that an increase in defecation in animals can constitute 
an emotional response. However, Archer, supra, noted that defecation 
decreases to normal levels in repeated testing and recommended that other 
behavioral data be considered before concluding emotionality is present. 
Another characteristic associated with pathological behavior useful for 
model selection is stereotyped behavior. Stereotypy is generally defined 
as senseless movements, actions, and/or words. Hall, C. S. Sigma XJ 
Quarterly 17-37 (1938) defined stereotypy as behavior characterized by the 
persistence of the same response in a free-choice situation and by a 
difference in the pattern of movement between an emotionally stable and 
unstable animal. For example, Munkvad, Acta Psycht. Scand. 191:193-199 
(1966) described sniffing, licking and repetitive cage biting as 
stereotypy exhibited by rats treated with monoamine oxidase inhibitor 
(MAOI) and then injected with 5HTP (a serotonin precursor). Unprovoked 
outbursts of violence, fast movement, and vocalization are also behavioral 
characteristics associated with psychosis which can be used for model 
selection. Generally, researchers classify this data under "rage 
reaction." See, Randrup, A. et al., Acta Psychiat Scand. SuppL 
191(42):193-199 (1966). 
In selecting appropriate animal models, it was once thought that abnormal 
behavior could be a consequence of parental care as opposed to an internal 
change, thus studies required cross-fostering as a control. However, 
cross-fostering experiments testing this hypothesis failed to show a 
difference in activity in the stressed and nonstressed offspring raised by 
their own mother. Hockman, C. H., J. Comp. Physiol. Psychol. 54(6) 679-684 
(1961). These results suggested reactions caused by postnatal separation 
experience (i.e. cross-fostering) as opposed to postnatal care. 
A common model of pathological psychotic behavior is drug-induced psychotic 
disturbance. However, the use of such models requires the Injection of 
drugs, such as amphetamines or morphine, to obtain the desired behavior. 
Moreover, drug-induced models are not entirely satisfactory for the 
desired psychotic disturbance due to the extremity of behaviors resulting 
from drug manipulation. For instance, in cats, a ten-fold higher dose of 
amphetamine is required to induce stereotyped behavior, than is required 
to awaken the cat. The high dosage required for drug-induced behavior also 
usually results in neurotoxicity which is undesirable in a model of 
psychosis. Moreover, drug manipulation may produce unavoidable 
stimulational effects on the animal due to the injection technique 
utilized (e.g. cutaneous, peritoneal, or intraventricular injection). 
Prenatal stimulation to change the uterine environment has been used to 
induce abnormalities (and thus possible models of psychosis) in offspring. 
For example, to add stress to the uterine environment during gestation, 
Fuchtgoh, E. et al., J. Comp. Physiol. Psychol. 51:541-545 (1958) applied 
x-irradiation, Thompson, W. R. Science 25:698-699 (1957) applied shock 
treatments, and Vincent, N. M. Am. Psychol. 13:401 (1958) administered 
alcohol to rats. Bogdanski, D. F. et al., J. Pharmacol. Exp. Therap. 
122:182-194 (1958) also noticed that 5HTP injection into non-pregnant 
animals produced a marked increase in uterine serotonin levels in cats and 
dogs. However, these studies did not report retained heritable effects in 
the F.sub.2 generation. 
With respect to retained heritable characteristics useful for the study of 
pathological behavior in psychotics, one animal model, the Maudsley rat 
strain, has been shown to exhibit high emotion in successive generations. 
The Maudsley rat was established by a classic selection scheme for 
behavior indicating high emotion and further in-breeding. Broadhurst, 
supra. The ability of selection by phenotype to produce a stable and 
reliable strain has been confirmed by the use of the Maudsley rat strain 
in experiments reported in over 300 publications. It is interesting to 
note that the Maudsley rat has abnormally high levels of brain serotonin, 
see Sudak, H.S . et al., Science 146:418-420 (1964). The genetic basis of 
the origin of the Maudsley rat has not, however, yet been defined. 
Sakuma, M., et al., Abstract No. 1974 in the 68th Federation Proceedings 
(1984) examined the effects of prenatal tryptophan diets on F.sub.1 
offspring, suggesting that behavioral changes may derive from the prenatal 
diet because the offspring with prenatal high tryptophan diets exhibited 
low activity, whereas the offspring with prenatal low tryptophan diets had 
high activity. However, the low and high tryptophan diet groups did not 
differ in any other quantifiable open field behaviors. For example, no 
statistically significant differences in defecation, spontaneous turning, 
flat walking and head shaking were observed, nor did either group display 
any rage reaction. Thus there was no indication that later generations 
would exhibit such pathological pyschotic behavior. 
The effects of tryptophan are of particular interest because tryptophan is 
used in various clinical applications, for example, as a painkiller, 
sleeping pill, bed-wetting control, menstruation discomfort reliever, and 
a sedative for depressive anxiety. However, it appears that tryptophan is 
not without adverse effects. For example, Mayeno, et al. Science 
250:1707-8 (1990) recently Identified the offender of Eosinophilia-Myalgia 
Syndrome (EMS), a malady associated with muscle pain, to be a combination 
of 1 -ethyltryptophan and tryptophan. Sakuma, M., Abstract, Society for 
Neuroscience (1992), postulates that EMS may be caused by combined 
influences involving tryptophan toxicity with conditions of genetic 
background and/or underlying metabolic abnormality as the predisposition. 
Therefore, further study of the effects of tryptophan use would be 
desirable. 
It would thus be desirable to provide an animal model for the study of 
pathological behavior relevant to psychotic syndromes. It would further be 
desirable to provide a simple method of production of such a model. It 
would also be desirable to have an animal model for the study of the 
genetic effects of tryptophan use. It would further be desirable to have a 
method for the breeding of heavier experimental animals and livestock. 
SUMMARY OF THE INVENTION 
The present invention provides an emotionally and behaviorally altered 
animal useful as a model of pathological behavior seen in psychosis. The 
animals of this invention were obtained by inbreeding mature F.sub.1 
offspring of rats wherein the mothers of the F.sub.1 received 3% (by 
weight) tryptophan supplemented diets during their third trimester of 
gestation. The present invention also relates to the preparation and use 
of animal models for the study of psychosis and the effects of tryptophan 
on successive generations. The effects of tryptophan treatment on 
successive generations can also be used to increase the body weight of 
experimental animals and livestock. 
The phenotypic attributes of these altered rats include behavior indicative 
of high emotionality, stereotypy and unprovoked rage reaction thus 
providing an appropriate congenital animal model for pathological 
behavior. Increased body weight of successive generations as a result of 
prenatal tryptophan treatment is also useful for breeding heavier 
laboratory animals and livestock. 
DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION 
Pregnant Sprague-Dawley rats artificially inseminated with Sprague-Dawley 
male sperm were obtained from Zivic-Miller Laboratories Inc., Allison 
Park, Pa. 15101. While in their third trimester of gestation (last week), 
the pregnant experimental rats received Pregnant Rat Tryptophan Deficient 
Meal available from Bio-Serv, Inc., (Frenchtown, N.J.) a corn powder diet, 
supplemented with three percent (3%) by weight tryptophan. On delivery 
day, the rats received regular Purina Lab Chow (0.3% tryptophan). The 
control group was fed the corn powder diet supplemented with only 0.3% by 
weight tryptophan during the last trimester of pregnancy. 
Although the F.sub.1 progeny of the tryptophan stimulated mothers exhibited 
a decrease in activity, no other signs of abnormality appeared. Several of 
the F.sub.2 generation, which was produced by inbreeding mature F.sub.1 
generation rats, exhibited a variety of behavioral abnormalities 
associated with emotionality. The precise mechanism of inheritance is not 
yet known. In part the mechanism of inheritance for animal behavior 
remains unsolved due to the complexity of gene activity on a behavior. 
These altered F.sub.2 rats showed less activity evaluated by low rearing 
counts and long stays in the corner. Also, these rats displayed a high 
rage reaction. Unprovoked, the rats shrieked and displayed aggressive and 
bizarre behavior. These rats also exhibited low activity by a short 
latency in corner reaching and a long latency in face washing. The body 
weight of these rats was also higher than that of control rats. Also, the 
rats showed stereotypy. 
These rats' emotional status demonstrated by the low activity, stereotypy, 
and rage reaction are useful for the study and therapy of psychosis. To 
confirm inheritance of targeted behavior, the characteristics of behavior 
are required to possess remarkable, distinguishable and 
glance-recognitionable behavior with persistent and frequent appearance. 
Among the behaviors of the animals of the present invention, the rage 
reaction and open-field behavior meets these demands. Furthermore, this 
method's effect of causing increased body weight can be used in the 
preparation and use of livestock. These rats and other animals produced by 
prenatal dietary intake of tryptophan can also be used in the study and 
therapy of tryptophan induced mutations. 
F.sub.2 rats, exhibiting the desired phenotype may be bred, cross-bred or 
in-bred and their progeny selected for the desired characteristics. In the 
alternative, F.sub.2 rats not exhibiting the desired phenotype may be bred 
and their offspring which exhibit the desired phenotype, if any, may be 
selected. In any event, once the desired phenotype is exhibited, 
successive generations can be obtained through natural breeding or 
artificial insemination and behavioral selection. 
For Specific Examples 1 through 3, Sprague-Dawley rats were used. There 
were 31 males and 28 females in the experimental group and 8 males and 13 
females in the control group. Observations were recorded at the ages of 3, 
6, 8 and 10 weeks in both the F.sub.1 and F.sub.2 generations. The 
specific examples are of the F.sub.2 generation of pregnant rats which 
received a 3% tryptophan diet in their last trimester of gestation. The 
parents (F.sub.1) of these rats were fed a normal diet containing 0.3% 
tryptophan.

SPECIFIC EXAMPLE 1 
Rage Reaction 
In general, experimental rats, such as the Sprague-Dawley, are highly 
inbred, docile and easy to handle and test, especially for the animal care 
person. The 3% tryptophan (H group) rats, however, exhibited extremely 
aberrant and aggressive behavior. 
One female and one male from two litters out of six litters of the H Group 
exhibited explosive pathological behaviors without stress or environmental 
change. For example, at ten weeks of age, during the open-field test, the 
female rat suddenly began a loud monotonous shrieking, then ran along the 
test box wall and jumped out from the box. Once out of the box, the female 
stood at the corner of the desk where the test box was placed, and 
continually shrieked and stared at the tester. The male rat showed even 
more aggressive and bizarre behaviors. For example, the male displayed 
abnormal characteristics by looking around as if in fear. Then, he 
screamed out loudly without provocation. While screaming, the rat snarled 
and bared his fangs at the tester. The behavior was characteristic of 
anger or a threat response. Finally, he rushed at his tester and leapt 
from the box. 
Open-field behavior is clearly influenced by genes at a variety of loci; 
however, the F.sub.2 progeny were produced by sibling mating which leads 
to progressive increases in homozygosity in each generation. Therefore, 
the F.sub.1 will be backcrossed to two parent generations (B1 and B2) to 
determine the phenotypic variations, i.e., additive, dominance, 
environment and maternal components. Also, reciprocal crosses will be 
performed to determine maternal effects and sex linkage for confirmation. 
SPECIFIC EXAMPLE 2 
Stereotyped Behavior 
Although the H group and the control group did not differ in the number of 
head shakes and body turns, the actual pattern of these behaviors did 
differ. Therefore, according to Hall, the rats displayed signs of 
stereotypy: the control group usually shook their heads side to side, 
whereas the H groups tossed their heads back repeatedly. The control 
group's body turning generally consisted of one or one and one-half turns 
while the H group's body would circle over three times in one spot. 
SPECIFIC EXAMPLE 3 
Open-Field Test Data (The data described below is in Table I which 
follows.) 
A. Rearing (exploration; standing on rear extremities). 
The 3% tryptophan group (H group) had lower scores than that of the control 
group (M group). So, using the Archer supra, criteria, both genders of the 
H group display abnormal open-field behaviors which are evaluated as high 
emotion. 
B. The Comer Crouch 
i. The first corner reach time (seconds) was measured as the time for the 
rat, starting from placement in the middle of the test box, to reach the 
test box corner, where the rat usually sits or crouches and does grooming, 
face washing, and sleeping. The male rats of the H group were quicker to 
reach the corner than male contol rats. 
ii. The corner crouching duration is the time measured while the rats 
remained in the corner and did not move around. The female rats of the H 
group had a long corner crouching duration which indicates low activity, 
i.e. apparently consistent with high emotionality. 
C. Face Washing 
i. The first facial wash is the time measured from placement in the middle 
of the test box, until the rat started the first face wash. Both male and 
female rats of the H group showed a delay before starting to face wash. 
ii. The facial wash counts have been reported but are combined with the 
grooming counts. This data is believed to show emotionality. 
D. Defecation 
The feces were counted for five minutes during the open field test. The 
females of the H group had significantly lower counts than the control 
group. 
TABLE 1 
______________________________________ 
Results of open-field test scores in F.sub.2 progeny (t-test). 
Control (M) vs. 3% TRP Group (H) 
Activity Male Female 
______________________________________ 
1st Rearing (seconds) 
ns ns 
Rearing Count M &gt; H M &gt; H 
6 wk, p = 0.1% 
3 wk, p = 1% 
6 wk, p = 2% 
1st Corner Reach (seconds) 
M &gt; H ns 
6 wk, p = 1% 
Corner Crouch Duration 
ns M &lt; H 
(seconds) 3 wk, p = 0.1% 
1st Facial Wash (seconds) 
M &lt; H M &lt; H 
3 wk, p = 1% 
3 wk, p = 5% 
8 wk, p = 5% 
10 wk, p = 0.1% 
Facial Wash Count 
ns ns 
Defecation (counts) 
ns M &gt; H 
10 wk, p = 5% 
______________________________________ 
M group: 8 males and 13 females 
H group: 31 males and 28 females 
ns = not significant 
p = probability 
SPECIFIC EXAMPLE 4 
Body Weight 
As shown in Table 2, both genders of the H group described in previous 
Examples show heavier body weight than that of the control group. Thus, 
prenatal tryptophan dietary treatment produces good growth even in the 
second generation. 
TABLE 2 
__________________________________________________________________________ 
Body Weight in Grams of F.sub.2 Progeny 
MALE Female 
AGE 3% TRP Group (H) 
Control (M) 
p 3% TRP Group (H) 
Control (M) 
p 
__________________________________________________________________________ 
3 weeks 
77.06 .+-. 15.7 
68.88 .+-. 9.1 
77.86 .+-. 18.8 
66.08 .+-. 10.3 
5% 
6 weeks 
214.90 .+-. 44.3 
185.50 .+-. 11.7 
181.75 .+-. 34.8 
161.38 .+-. 11.76 
8 weeks 
344.29 .+-. 32.9 
308.38 .+-. 17.3 
1% 
245.64 .+-. 23.2 
214.15 .+-. 12.5 
0.1% 
10 weeks 
411.29 .+-. 47.7 
383.13 .+-. 22.2 
278.54 .+-. 26.9 
246.92 .+-. 13.5 
0.1% 
__________________________________________________________________________ 
M Group: 8 males and 13 females 
H Group: 31 males and 28 females 
p = probability 
Those skilled in the art can now appreciate from the foregoing description 
that the broad teachings of the present invention can be Implemented in a 
variety of forms. For example, other species of animals can be employed. 
In some circumstance, for instance, it may be desirable to use a species, 
e.g., a primate such as the rhesus monkey, which is evolutionarily closer 
to humans than rats. Moreover, although a ten-fold dietary increase in 
tryptophan (0.3% to 3.0% by weight) was used in the preferred embodiment 
described herein, the present invention includes other increased levels of 
tryptophan which are sufficient to produce the desired heritable phenotype 
without undue toxic side effects. Therefore, while this invention has been 
described in connection with particular examples thereof, the true scope 
of the invention should not be so limited since other modifications will 
become apparent to the skilled practitioner upon a study of the drawings, 
specification and following claims. 
All publications cited herein are incorporated by reference.