maze had a start and goal box at opposite ends
of a square configuration wherein removable bar-
riers of varying lengths could be arranged .in
such a manner as to provide twelve different
maze tasks, of which Problems 1, 8, 5, and 11
were used.2? A wire-mesh screen top provided
E (experimenter) with an unobstructed view of
the entire maze from a wooden cubicle (42’” x
72/’ x 42/’) with a one-way vision glass insert.
This arrangement also prevented possible / dis-
traction for the Ss. A chair, built-in counter in
the cubicle, stop watch, and scales for weighing
the animals completed the test equipment.

Procedure. Since there were too many ani-
mals to run in a single session, each group of 10
was subdivided randomly into three smaller
groups of four, three, and three rats. The first
animals tested consisted of four controls, four of
the 10-p.p.m. group, four of the 25-p.p.m. group,
and four of tho 50-p.p.m. group. These were
run in random order. In all cases the reward
was the food to which they were accustomed.
Thus, the control group received unpoisoned
food, the 10-p.p.m. group received food contain-
ing 10-p.p.m. of poison, etc. Testing and train-
ing of the subgroup of four began after 13 weeks
of exposure, and of the subgroups of three each
after 14 or 15 weeks.

Prior to each familiarization and test session,
all animals were weighed to ensure that rela-
tively equal body-weight losses were maintained.
This procedure was used to minimize possible S
differences in drive as a function of body weight.
All Ss received five familiarization and five test
trials on each of the four separate maze problems
in a sequential manner (ie., Rabinovitch and
Rosvold’s® problems 1, 3, 5, and 11, in that order)
with familiarization and test trials given on
alternate days, i¢., all Ss in each of the four
groups were given familiarization trials on prob-
Jem No. 1 one day, and five test trials on the
same problem the following day, at approxi-
mately the same time of day the familiarization
trials were administered. This procedure was
followed until four diferent maze problems had
been presented. Alternating days for familiari-
zation and test trials were employed as a means
of reducing performance fatigue and/or food
satiation effects. Prior checks indicated that
most of the initial learning effects occurred dur-
ing the first five familiarization trials. It should
be noted that since prior experience may have

cortical or subcortical ACh activity effects,> all
animals received identical treatment during all
phases of the experiment.

Scores for a trial consisted of the number of
errors made and the length of time taken by a S
in traveling from the start box to the goal box.
Errors were counted as the number of wrong
turns including retracing maneuvers. Upon en-
tering the goal box the S was permitted to feed
for 80 seconds as reward, after which he was
replaced in the start box. All Ss were main-
tained at 15 to 20 percent bodyweight loss during
all phases of the experiment.

Performance measures consisted of the mean
number of errors made and mean trial times for
a five-trial session for each of the four problems
presented. Immediately prior to sacrifice, all Ss
were weighed for a final time. Following sacri-
fice, whole-brain assays were made.*

Ill. Results.

AChE levels for the 10-p.p.m., 25-p.p.m., and
50-p.p.m. treated groups showed statistically sig-
nificant reductions of 59 percent, 67 percent, and
74 percent, respectively, with the mean AChE
level of controls representing zero reduction.
There was, however, no statistically significant
difference in the amount of reduction among the
poisoned groups. All AChE levels were near or
beyond the 60 percent critical reduction level
considered necessary to severely affect ACh con-
tent... Means and standard deviations of the
number of errors and trial times for each group
in each of the four test sessions are shown in
Table 1.

An analysis of variance test of errors’ indi-
cated significant between-group differences as a
function of poison; however, no differences were
found to be present as a function of the four
maze problems (probably attributable to the five
practice trials each animal had in each maze
prior to the second performance trials, and to
the order of maze presentation, i.e., all animals
began with the least difficult maze), nor were
interaction effects present. (See Table 2.)
Orthogonal comparisons revealed significant dif-
ferences between the controls and the 10-p.p.m.
Ss (F=102.95, p<.001), the 25-p.p.m. Ss (F=

*We wish to thank Mr. Leonard C. Ryan of CAMI’s
Pharmacology-Biochemistry Laboratory for performing
these assays.



crease in exposure level may well bring overt
symptoms which might mean disaster for an
agricultural pilot.

Moreover, with chronic exposure to organophos-
phates and chronic lowering of AChE, the margin
of safety is markedly lessened and a slight in-

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7.

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NacuMAnsoun, D.: Chemical and molecular basis of
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3. Bennerr, E, L., Dramonp, M, C., Kreck, D., and 18 McIsaac, R, J. and Koxrrx, G. B.: Comparison of
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39-82, 1963.


animal in a maze under apparently less traumatic
conditions.

IL. Method.

Subjects. Forty male Charles River rats
(albino) approximately 30 days old were ran-
domly assigned in equal numbers to a control
condition or to one of three level-of-poison con-
ditions immediately upon arrival at the labora-
tory. Each group was housed separately and
fed a powdered form of a normal diet food.
The experimental groups received either a 10-
p-pm., 25-p.p.m., or 50-p.p.m. addition of the
AChE inhibitor Disulfoton (O, O-diethyl S-2-
(ethylthio) ethyl phosphorodithioate), a semi-
reversible organophosphorus AChE inhibitor,
mixed in with their normal diet of ground-up
pellet food. Both food and water for the next 3
months were available ad libitum. Since our ani-
mals received the poison, Disulfoton, in their
food, the trauma of repeated intramuscular or

intraperitoneal injections was obviated. Also,
our experimental rats gained weight on the diet
(until, by design, a weight loss was introduced to
ensure a hunger drive) which indicates that long
before training began the rats were well into the
tolerant phase.® There were thus two distinct
advantages to our procedure. First, since neither
injections nor intubations were made, the rats
could not associate removal from their home
cages with pain which made them, perhaps, more
amenable to handling. Secondly, the experiment
was more comparable to the situation in man for
we were not interested in ‘acute effects, but rather
with the long-term chronic effects such as might
occur in agricultural pilots.

Other, similarly poisoned animals were used
to determine the amount of weight-loss that
could be used to assure motivation (food search-
ing) without damaging the rats.

Apparatus. A  closed-field intelligence test
maze for rats* was used (see Figure 1).. The

Ficure 1. The closed-field maze arranged for presentation of Rabinovitch and Rosvold’s “Problem 5.”


Tasie 1.—Number of errors and trial times in each test

session for the control and Disulfoton-exposed groups.

Mean Mean

Test session Group number of sD trial time sD
errors (min.)

2.6 1,07 2.33 2.16

1.8 1.31 0.54 0.42

1.8 1,68 0.35 0.33

17 1.25 0.45 0.37

3.1 1,28 1.59 1.18

2.2 1.08 0.24 0.14

1.6 0.84 0.36 0.31

50-p.p.m. 1.6 0.84 0.20 0.12

3.0 1,49 1.37 1,09

Lt 0.73 0.21 0.13

. 14 1.17 0.20 0.14

50-p.p.m. 0.9 1.10 0.16 0.14

ee Control 3.8 2.14 1,28 1,16

10-p.p.m. 15 1,26 0.51 0.47

25-p.p.m. 14 0.69 0.26 0.30

50-p.p.m. 14 0.84 0.46 1.16

117.42, p<.001), and the 50-p.p.m. Ss (F=140.85,
p<.001). Additional orthogonal comparisons
revealed no significant performance differences
between the groups with reduced AChE. An
analysis of variance test of the “trial times”
data indicated significant group differences as a
function of poison. Orthogonal comparisons re-
vealed that the significant differences in mean
trial times were between control animals and
each of the 10-p.p.m. (F=107.19, p<.001), 25-
ppm. (F=126.35, p<.001), and 50-pp.m. (F=
120.44, p<.001) groups. (See Table 3.) Similar
orthogonal findings of error scores for the
between-exposed-groups were obtained for the
trial times. Again, no significant differences
were found as a function of the four maze
problems.

Tasue 2—Summary analysis of variance of the effects
of various levels of chronic Disulfoton exposure on
number of incorrect responses.

Source df MS F

3
Sessions (8) - 3
GxS.-- 9
Error (G)- 36
Error (S plus @x 8)... 108

*p <.001

‘TasLe 3.—Summary analysis of variance of effects of
various levels of chronic Disulfoton exposure on trial times.

Source af MS F
Groups (G)- - 3 9.62 —*14,80
Sessions (S)- - 3 1.17 -99
GxS.. - 9 -58 249
Error (G)- 36 65 _..
Error (8 plus G x §)- 108 1.18.2.

*p <.001

IV. Discussion.

Organophosphates are inhibitors of cholin-
esterase, which is considered necessary in pre-
venting excessive acetylcholine accumulations.
It is generally thought that excessive accumula-
tion of ACh leads to hyperactive and/or irregular
synaptic transmissions at all cholinergic sites so
affected. Thus, it is possible that the central
processes of those routinely exposed to AChE may
be detrimentally affected, resulting in measurable
performance impairment. The results of this
study indicate that rats fed relatively high
amounts of Disulfoton were capable of perform-
ing a task with fewer errors and shorter trial
times than their controls, although the AChE
reduction in the most severely exposed group
was more than 75 percent of normal. These re-


AM 69-19

A COMPARISON OF THE BEHAVIORAL EFFECTS OF
VARIOUS LEVELS OF CHRONIC DISULFOTON POISONING

David W. Pearson, Ph.D.
George Clark, Ph.D.

Carl M. Moore
Approved by Released by
; Wb, As PY S2 7 v7 »)
J. Ropert Diniz, M.D. P. V. Srecen, M.D.
Cuter, Cry, ArRoMEDICAL Fepera Air Surcron

Institute

October 1969

Department of Transportation
FEDERAL AVIATION ADMINISTRATION
Office of Aviation Medicine



sults obtain, however, only in the event that
physiological effects do not inhibit the behavior
under investigation, ic. if running time in a
maze is the dependent variable, then the animal
must be able to run. For example, inhibition
effects occurred twice during familiarization
tests (i.e. one 50-p.p.m. S had an epileptic type
seizure and another 50-p.p.m. S had body tremors
to the degree that walking became impossible).
These latter effects also suggest that lethal dose
levels were approached for the 50-p.p.m. group.
It should be stressed that, except in these latter
two instances, the 50-p.p.m. group (with brain
AChE reduced to less than 75 percent of normal)
was able to perform as well as the 10-p.p.m.
group (with brain AChE reduced to 59 percent
of normal) and significantly better than the
controls. Differences in errors or trial times be-
tween the Disulfoton-exposed groups were not
found and may have been due to the fact that the
AChE reductions for the 10-p.p.m., 25-p.p.m.,
and 50-p.p.m. animals were near or below the
60 percent: reduction level considered by Aprison*
as necessary to critically affect ACh content.

In some respects the results reported here are
different fromi those obtained by other investi-
gators. Thus, Merrill** found no time-to-solution
differences between albino mice that had been
poisoned at various dose levels and controls (the
poison presumably resulting in various AChE
reductions) in a place learning task. Glow, et
al.7° using female Wistar rats, reported signifi-
cant differences in time to obtain 20 reinforce-
ments between AChE-reduced groups and their
controls, ie., the AChE-reduced animals per-
formed slower than the control animals. Glow,
et al? report, however, that their results were
obtained, in part, as a result of the immediate
effects of booster injections although “these slow-
ings in the press rate became attenuated with
each successive injection” (p. 156). Addition-
ally, Merrill*® reported that both the highest and
lowest AChE-reduced groups made significantly
more errors than the middle-range of AChE-
reduced groups or the control animals. This is,
in part, in contrast: with the findings of this study
which showed significantly fewer errors by the
AChE-reduced groups than the controls.

Since other evidence indicates that ACh has
manifold functions in addition to that of synaptic
transmitter? and that AChE can exist at both
functional and reserve levels,* much more infor-

mation is needed, in addition to behavioral data,
to suggest any theory (e.g., an increased general
activity drive) which might explain the manner
in which the lowered AChE levels in these rats
resulted in fewer errors and more rapid traver-
sion of a maze task, or to extrapolate any of the
findings reported above to human subjects. The
fact that the performance of Disulfoton-treated
animals was facilitated in this study may be
contrasted with the physiological changes (eg.,
seizure and incapacitation) induced by the chem-
ical treatment in two animals from the most
severely exposed group.

Hebb“ in his discussion of “Emotion and
Motivation; the Social Context” states that, in
general, stimuli at lower levels of intensity pro-
mote waking up and, at progressively higher
levels, increasing alertness and, finally, disor-
ganization. Similarly, intoxication with ethanol
can seriously impair performance, while lower
levels may cause no decrement,® or even an im-
provement in behavior.t Coffee and other ex-
ternal or mental stimulants all may increase the
level of excitation and of activity and, up to a
certain level, may produce an apparent improve-
ment in behavior, while higher levels may pro-
mote disorganized behavior and decreased per-
formance. Our rats, which were exposed to
critically high amounts of Disulfoton, showed
not only no performance decrement, but a sta-
tistically significant increase in performance so
long as the animals were not incapacitated by
tremors or convulsions. The extent to which
these results may be applied to man is not known
although there are some indications in the litera-
ture that, in man, low blood AChE levels are not
necessarily associated with decreased perform-
ance. This can be seen in the reports of Sumer-
ford, Hayes, Johnston, Walker, and Spillane,”
of Gershon and Shaw,! of Dille and Smith’ and
of Smith, Stavinoha, and Ryan." However, the
possibility of either antagonism or synergism
with a combination of insecticides is very real
and this interaction should be investigated.

Nore.—While the albino Charles River rats used
in this experiment have very limited or no vision
and thus the stimuli used by the animals in tra-
versing the maze were not visual, we have replicated
the experiment (one problem only) with Long Evans
rats. These hooded rats have excellent vision. The
results with the Long Evans rats were identical to
those with the Charles River Strain.



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A COMPARISON OF THE BEHAVIORAL EFFECTS OF VARIOUS
LEVELS OF CHRONIC DISULFOTON POISONING

I. Introduction.

In jobs related to aerial application of toxic
pesticides, there exists the danger of accidental
exposure, Such exposure of general aviation
pilots has been reported as a possible source of
impairment of flying performance; in some in-
stances exposure of pilots to pesticides may have
contributed to fatal crashes.” Of particular
concern are the organophosphates that are known
to be brain AChE (acetylcholinesterase) inhib-
itors. It is generally believed that, if ACh
(acetylcholine) is one of the most important
transmitter substances in the central nervous
system, it would seem almost assured that severe
alterations in cholinesterase (which is responsible
for the rapid destruction of ACh)** would mark-
edly affect the function of ACh as a transmitter
agent. Thus, it might be thought that signifi-
cant changes in AChE levels would affect such
processes as memory, learning, and/or similar
mental performances.

Although there are several reviews and reports
on anticholinesterases and their physiological and
pharmacological significance,**° few formal
studies have been concerned with the behavioral
consequences of cholinesterase inhibition. Of the
animal studies on the effects of AChE inhibition,
a large number deal with the acute stages of
AChE reductions. Many of these report only
the immediate toxic reactions of the organism
such as fasciculation, tremor, diarrhea, etc.,
rather than the interjective effects upon an on-
going conditioned response” after administration
of an AChE inhibitor" * Further, in all of the
studies of acute poisoning, injection or intubation
techniques were used. The Ss (subjects) were
given rather large doses of an AChE inhibitor
in a relatively brief period of time; and with the
exception of a study by Russell, Watson, and
Frankenhaeuser,® all of the chronic studies
(AChE reductions maintained over a period of
time) used similar techniques.” ° ** **

Most of the acute and chronic AChE inhibition
studies noted above generally reported no differ-
ences in learning abilities between Ss with re-
duced AChE activity and control Ss, but they
did find differences in rate of extinction of a
conditioned response, ie, Ss with severely re-
duced AChE activity seem to take a longer period
of time to extinguish a response than do control
Ss. Although injection techniques are commonly

- used in both chronic and acute studies, equivocal

and, in many cases, contradictory results are re-
ported. For example, Glow and Richardson?
investigated the behavioral effects of AChE re-
ductions on both selective and nonselective sites.
They reported no differences in rate of extinction
of poisoned versus control Ss in selective-site
investigation, i.e., reductions restricted to the
brain, ‘which was in contrast, to the effects “of
an acute, ‘nonselective’ reduction in AChE activ-
ity, Le., reduction throughout the body” (p. 430).
Natoff?” states that one reason for these dissimilar
results may be due to the fact that the adminis-
tration of drugs to animals via different methods
or routes can produce different results, even to
the same strain of animals. Despite a growing
consensus concerning the importance of using a
noninjective technique, i.e., one more analogous
to the natural feeding habits of animals, many
investigators continue to ignore this problem.
As indicated earlier, only one study reported
the effects of both chronic and acute AChE re-
duction upon performance. Glow, et al.1° sug-
gested that any differences noted between Ss with
reduced AChE activity and control Ss may have
been due entirely to “the malaise brought on by
the treatment,” ie, the administration of
“booster” injections to maintain a chronic AChE
reduction level may have produced adverse
physiological effects. Thus, the present study
was designed to explore the extent to which
chronic AChE inhibition at various levels of
reduction might affect the performance of an