canging from 0.025-0.400 mg/kg. The animal
was closely observed at all times through a
“one-way” window in the isolation chamber.

Six experiments were also done in pentobarbi-
al anesthetized monkeys held in a stereotaxic
ypparatus. Qualitatively, the results were iden-
sical to those seen in the chronic preparations.

III. Results.

Figure 1 shows a “typical” septo-hippocampal
AER. Amplitudes of response peaks were meas-
ured. The primary negative wave (d) of the
AER was most sensitive to mevinphos action and
all future references to AER amplitudes will
refer to wave (d).

ACUTE PHOSDRIN EFFECTS

120

4 _

jo)

ing

- OF

2

°

2 100

z

a "05

‘J 90 nl 10

= ol

> 80f “

5 PHOSDRIN I.M. DOSE
70oF

5

L L l l l 1

a
120 150 180 210 240 330
TIME (MIN.)

! !
30 60 90

eure 2. A plot of the effects of mevinphos on hippocampal evoked potential amplitudes. For the calculation of

these points, the pre-drug control values in each run were averaged. This reference average value was taken
as 100%. All potential amplitudes were then plotted as a percentage of this reference average, which is rep-
resented by the single dot in the dotted band. The extreme variation of the pre-drug control amplitudes was
within 5% of the reference average. This zone of variation is represented by the dotted zone in the figure.
AEP peak values occurring within 5% of the reference average were not considered significant and were not
plotted. Pre- and post-drug AEP amplitudes were also tested with the Mann-Whitney U-test. The plotted
points were all significant at the 0.05 level. In non-drug control studies the AEP amplitudes always remained
within the 5% deviation band. Each plotted point above represents the means of 3 to 5 tests. As can be seen
the administration of mevinphos induces an inhibition of AEP amplitudes. The extent and duration of this
inhibition are proportional to dose within this tested dose range.

3



REFERENCES

1. Adey, W. R.: Recent Studies of the Rhinencephalon
in Relation to Temporal Lobe Epilepsy and Be-
havioral Disorders, INT. REV. NEUROBIOL,
1:1-48, 1959.

2. Anderson, P. and Lomo, T.: Mode of Control of
Hippocampal Pyramidal Discharges. In: The Neural
Control of Behavior, edited by R. Whalen and R.
Thompson, New York, Academie Press, 1970.

3. Auerbach, A. A.: Transmitter Release at Chemical
Synapses. In: Structure and Function of Synapses,

GD. Pappas and D. P. Purpura, editors, Raven,
New York, 1972.

. Biscoe, T. J, and Straughn, D. W.: Micro-Electro-
phoretic Studies of Neurones in the Cat Hippo-
campus, J. PHYSIOL, 183 :341-359, 1966,

5. Blackstad, T. W.: On the Termination of Some
Afferents to the Hippocampus and Fascia Dentata,

ACTA ANAT., 16:202-214, 1958.

6. Blakemore, C., Iversen, S. D., and Zangwill, 0. L.:
Brain Functions, ANN. REV. PSYCH., 23:413~456,
1972.

. Fink, Max: EEG and Human Psychopharmacology,
ANN, REV, PHARMACOL., 9:241-258, 1969.

8 Friede, R. L.: Topographic Brain Chemistry, Aca-

demic Press, New York, Chapter X :237-281, 1966.

9. Green, J. D,: The Hippocampus, PHYSIOL. REV.,
44::561-608, 1964.

10. Hayes, Wayland J.: Pesticides and Human Toxicity,
ANN. N. Y. ACAD. SCI, 160:40-54, 1969.

11. Heath, D. F.:Organophosphorus Poisons, Pergamon
Press, New York, 1961.

12. Izquierdo, I, and Izquierdo, J. A.: Effects of Drugs
on Deep Brain Centers, ANN. REV. PHARMACOL,
11:189-208, 1971.

13. Koelle, George B.: Significance of Acetylcholinest-
erase in Central Synaptic Transmission, FED.
PROC., 28(1) :95-100, 1969.

»

a

14. Lewis, Mark F., Mertens, Henry W., and Steen, Jo
Ann: Behavioral Changes from Chronie Exposure
to Pesticides Used in Aerial Application: Effects of
Phosdrin on the Performance of Monkeys and
Pigeons on Variable Interval Reinforcement Sched-
ules, FAA Office of Aviation Medicine Report No.
AM-72-29, 1972.

15, Lewis, P. R. and Shute, C. ©. D.: The Cholinergic
Limbie System: Projections to Hippocampal Forma-
tion, Medial Cortex, Nuclei of the Ascending Cholin-
ergic Reticular System, and the Sub-fornical Organ
and Supra-optic Crest, BRAIN, 90:521-540, 1967.

16. McLardy, T.: Hippocampal Formation as Detector

Coder of Temporal Patterns, PERSPECTIVES
BIOL. MED., 2 :443--452, 1959.
17. Manocha, Sohan L., and Shantha, Totada R.:

Macaca Mulatta, Enzyme Histochemistry of the
Nervous System, Academic Press, New York, 1970.

18. Metcalf, D. R., and Holmes, J. H.: EEG, Psycho-
logical and Neurological Alterations in Humans with
Organophosphorus Exposure, ANN. N. ¥. ACAD.
SCL, 160 :357-365, 1969.

19. Namba, T., Greenfield, M., and Grob, D.: Malathion
Poisoning, ARCH. ENVIRON. HEALTH, 21:533-541,
1970.

20. Natoff, I. L.: Organophosphorus Pesticides: Pharma-
cology, PROGR. MED. CHEM., 8:1-87, 1971.

21. Pradhan, S. N., and Dutta, 8. N.: Central Cholin-
ergiec Mechanisms and Behavior, INT. REV. NEURO-
BIOL, 14:173-281, 1971.

22. Segal, M., and Olds, J.: Behavior of Units in Hippo-
campal Circuit of the Rat during Learning, J.
NEUROPHYSIOL, 35 :680-690, 1972.

28, Stumpf, (.: Drug Action on the Electrical Activity
of the Hippocampus, INT. REV. NEUROBIOL,
8:77-138, 1965.

24. Werman, R.: CNS Cellular Level:
REY. PHYSIOL., 34 :337-374, 1972.

Membranes, ANN.


SEPTO-HIPPOCAMPAL EVOKED POTENTIAL

Ficvre 1.

successive evoked potentials taken at one second intervals.
corder and this figure is a slightly smoothed redrawing of the X-Y recorder tracing.
Waves (c) and (d) represent predominantly excitatory potentials and are conventionally terme¢

artefact.

e

This is a recording of a septo-hippocampal averaged evoked potential.

100pn V.

10 ms.

This AEP was formed from 10C
The computer output the AEP onto an X-Y re
Wave (a) is the stimulus

negative waves, since that is the sign of the response at the level of the hippocampal pyramidal cell bodies
The subsequent waves represent intrahippocampal inhibitory phenomena and other secondary transmission pro
cesses, and wave (b) represents activity in the incoming nerve fibers.

hippocampus. Similarly configured stimulating
electrodes were placed in right dorsal hippo-
campus, right lateral septal nucleus, right lateral
amygdalar nucleus and the left side dorsal and
ventral hippocampus. By use of this electrode
array a variety of independent mono- and poly-
synaptic inputs to hippocampus could be acti-
vated?°°® so that any tendency of the OPs to
selectively affect hippocampal inputs could be
evaluated. After placement, the electrodes were
fastened to the calvarium of the monkey with a
system of screws and dental repair acrylic, and
were connected to the contacts of an ELCO
socket also anchored in the dental acrylic mass.
After recovery from surgery, the monkey showed
perfectly normal behavior, although it could at
any time be connected to our apparatus for
evoked potential studies.

For these experiments, the animal was placec
in a conventional 2-plate restraint chair ir
an isolation chamber. Amygdalo-hippocampal
septo-hippocampal and/or _ trans-hippocampa
evoked potentials were generated with Tektroni>
pulse generators. The amplified responses wert
sent to the LINC-8 computer which formec
averaged evoked responses (AERs) and storec
them on tape for later analysis, Animals wer
examined daily. AERs were taken at 15 minut:
intervals. After the third AER, the animal wa:
given an intramuscular injection of 10ul of fluic
per kilogram body weight. The injected fluic
was normal saline either by itself or containiny
mevinphos. AERs were then taken for a furthe
300 minutes, and the animal was returned to hi
cage. For these acute studies mevinphos wa
administered no more than once weekly at dose


Doses of mevinphos below 0.050 mg/kg pro-
duced no AER changes. Doses of 0.40 mg/kg
produced hippocampal seizures (although gen-
eralized CNS seizures were not usually seen),
together with marked peripheral signs of OP
poisoning (i.e., salivation, pupillary constriction,
tremor, weakness, etc.). Post-ictal AERs were
not taken since hippocampal seizures alter hippo-
campal bioelectrogenesis for up to 24 hours
(Revzin & Costa, unpublished observations;
Gergen & McLean, personal communication).

Mevinphos, in doses of 0.050 to 0.200 mg/kg,
caused an inhibition of the AERs (Figure 2).
The peak inhibition was dose-related and ranged
from 7% at 0.050 mg/kg to 28% at 0.200 mg/kg.
The duration of the effect was also dose-depen-
dent and ranged from 45 to 180 minutes. As
seen in Figure 2 the variance was quite small,
even in this pooled data from all animals. Inhi-
bition was seen in all components of the AER
and in all AERs studied—although drug effects
tended to be more marked on the septo-hippo-
campal response.

No peripheral signs were seen at dose levels
of 0.05 to 0.20 mg/kg, although two animals
developed brief (2-3 Sec) hippocampal seizure
episodes after 0.20 mg/kg. That is, there was
no observable tremor, weakness, pupillary con-
striction, or salivation, As mentioned, such
signs were seen at doses above 0.20 mg/kg.

IV. Discussion.

Microiontophoretic and other neuropharma-
cological studies have demonstrated that there
are powerful excitatory cholinergic receptors on
or near the pyramidal cell somata in hippo-
campus.* ** Therefore, acetylcholine will cause
depolarization of hippocampal pyramidal neu-
rones, and nerve endings in their vicinity.‘ ?
The negative wave of the evoked response (ER)
measured in this study is associated with excita-
tion of nerve cells? That is, peak (d) of the
ER is predominantly the vector sum of excita-
tory post-synaptic potentials (epsp’s). Now, the
amplitude of the epsp and the amount of trans-
mitter released from the nerve ending by each
impulse are both directly proportional to the
absolute magnitude of the membrane potential
of the neuron soma or ending.*** Since mevin-
phos is an AChE inhibitor it will increase the
concentration of free ACh in hippocampus.’*
This free ACh will depolarize endings and cell

bodies. Thus mevinphos should, as we have
shown, decrease hippocampal evoked response
amplitudes both by reducing epsp magnitudes
and by reducing the total amounts of transmitter
released by stimulation. ACh also increases
membrane conductance‘ ** in hippocampal neu-
rones, and this, in effect, partly short-circuits all
post-synaptic potentials in hippocampus.‘ ** This
effect. also contributes to the reduction of ER
amplitudes produced by mevinphos. Since these
effects are generalized effects on nerve cell mem-
brane, all hippocampal ERs may be expected to
be affected-—-as was found. Furthermore, the
cholinergic synapses in hippocampus originate in
septal neurones and through-fibers from midbrain
running through the septum.** * Therefore, one
would expect that the septo-hippocampal ER
would be most affected by mevinphos since the
cholinergic nerve endings and associated post-
synaptic membrane would, presumably, be ex-
posed to the highest concentrations of free ACh.
Though there was a tendency in this direction,
the differences between the septo-hippocampal
ER inhibition and the others were not significant.
There is no conclusive explanation for this dis-
parity. However, the cholinergic fibers are only
a small percentage of the nerve elements which
can be actuated by septal stimulation.s® Thus,
even dramatic changes in evoked activity at
cholinergic sites might be partly masked by other
events going on at the same time.

Obviously, if the depolarization caused by
ACh is sufficiently marked, the neurones will
begin spontaneous firing and seizure activity can,
result. Biscoe and Straughn reported this in
their microiontophoretic experiments‘ and it is,
probably the explanation for the hippocampal
seizures seen after mevinphos doses of 0.20 mg/kg
or more. As usual in hippocampal seizures, the
ictal activity did not propagate beyond hippo-
campus.1?° Thus, our results are in full accord
with what is known of cholinergic functions in
hippocampus.

Dr. Lewis and co-workers have reported quan-
titatively similar data on the effects of mevinphos
on behavior in the squirrel monkey. That is,
they reported behavioral inhibition over a dose
range of 0.050-0.250 mg/kg, the duration and
severity of the inhibition being dose-dependent.
The parallelism between their behavioral data
and the present electrophysiological findings is
striking and, perhaps, suggests the possibility


TECHNICAL REPORT STANDARD TITLE PAGE

1. Report No. 2. Government Accession No. 3. Recipient's Catalog No.
FAA~AM-73-3 —
4, Title and Subtitle . Report Date
February 1973
SUBTLE CHANGES IN BRAIN FUNCTIONS PRODUCED BY SINGLE ebruary
DOSES OF MEVINPHOS (PHOSDRIN) 6. Performing Organization Code
7. Author!s) B. Performing Organization Report No.

Revzin, Alvin i Ph.D.
9. Performing Organization Name and Address 10. Work Unit No-

FAA Civil Aeromedical Institute We San
P. 0. Box 25082 ~ Contract or Grant No.
Oklahoma City, Uklahoma 73125

13. Type of Report and Period Covered

12, Sponsoring Agency Name ond Address

Office of Aviation Medicine
Federal Aviation Administration
800 Independence Avenue, $.',

Washington, 0,C, 20591
5. Supplementary Notes

14. Sponsoring Agency Code

This work was performed under Task No, AM=Be73eTOve15
6. Abstract

Mevinphos (Phosdrin) was found to inhibit the amplitude of hippocampal evoked
potentials in unanesthetized squirrel monkeys with chronically indwelling
electrodes, ‘The threshold dose was 0,059 mg/ke and the maximal dose studied
was 0.200 mg/kg. Doses above 0.200 ma/kg induced hippocampal seizures, Within
the dose range of 0,950 ma/kg to 9,200 mg/kg the amplitude and duration of the
inhibition were directly proportional to dose, No peripheral signs of poisoning,
such as tremor or salivation, were seen at doses of 9.200 mg/kg or under, The
discussion emnhasizes that mevinphos produces changes in brain function in the
absence of the perinheral symptomatology usually taken as indicators of
poisoning by aerial applicator personnel, Therefore, it is concluded that
exposure to mevinphos may be unexpectedly hazardous since the aerial applicators
may be unaware that they have been poisoned,

7. Key Words 18, Distribution Statement

Anticholinesterase Acute Availability is unlimited, Document may
Pesticide Mevinphos be released to the National Technical
iippocampus Phosdrin Information Service, Springfield,

ivoked Potentials Aerial Applicators | Virginia 22151, for sale to the public.
Squirrel Monkey

5. Security Classif. (of this report) 20. Security Classif, (of this page) TI. No. of Pages | 22. Price
Inclassified Unclassified 6 $3,990

vm DOT F 1700.7 (8-69)



of a causal relationship between the changes in
hippocampal potentials seen here and their be-
havioral inhibition. As in the present study, no
peripheral symptomatology was seen at doses of
Jess than 0.250 mg/kg.

Perhaps the most noteworthy finding was that
mevinphos could induce hippocampal biopoten-
tial and behavior'* changes—including seizure
activity—without any of the usual peripheral
manifestations of OP poisoning. This is a bit
surprising, and disquieting. Although mevin-
phos is said to penetrate the blood-brain barrier
more readily than most other OP pesticides, it
does not penetrate freely.2 7° Thus, one would
expect that peripheral effects would tend to de-
velop before the CNS effects of the OP. That
this order is reversed suggests that the effects of
mevinphos on AChE in the CNS differ from
those in the periphery and/or that mevinphos
has actions in the CNS not directly related to its
effects on AChE.

More importantly, the data suggest that aerial
applicator personnel exposed to mevinphos—and,
perhaps, the other OPs—can suffer significant
CNS dysfunctions, even local hippocampal seiz-
ures, in the absence of the usual “peripheral”
pathognomonic signs. Since hippocampus “plays
a crucial role in the programming of acquired
sensory-response patterns,”* the hazards to the
aerial applicator may be substantial in absolute

terms, the more so since patients seem generally
unaware of hippocampal dysfunctions, even
though substantial deficits in performance, con-
sciousness or memory may be present.?® As a
practical matter these data reinforce previous
emphasis on the need for extreme caution in
handling the organophosphate pesticides.

Vv. Summary.

Mevinphos (Phosdrin) was found to inhibit
the amplitude of hippocampal evoked potentials
in unanesthetized squirrel monkeys with chron-
ically indwelling electrodes, The threshold dose
was 0.050 mg/kg and the maximal dose studied
was 0.200 mg/kg. Doses above 0.200 mg/kg
induced hippocampal seizures. Within the dose
range of 0.050 mg/kg to 0.200 mg/kg the ampli-
tude and duration of the inhibition were directly
proportional to dose. No peripheral signs of
poisoning, such as tremor or salivation, were seen
at doses of 0.200 mg/kg or under. The discussion
emphasizes that mevinphos produces changes in
brain function in the absence of the peripheral
symptomatology usually taken as indicators of
poisoning by aerial applicator personnel. There-
fore, it is concluded that exposure to mevinphos
may be unexpectedly hazardous since the aerial
applicators may be unaware that they have been
poisoned.





SUBTLE CHANGES IN BRAIN FUNCTIONS PRODUCED BY SINGLE
DOSES OF MEVINPHOS (PHOSDRIN)

I. Introduction.

Although the organophosphate _ pesticides
(OPs) have been extensively studied,!° 1 15 15 19 20
many areas of prime significance to aerial appli-
cator personnel remain unexplored. For example,
OP poisoning generates a complex mix of symp-
toms originating in part from effects on pe-
ripheral cholinergic synapses and in part from
effects on the central nervous system (CNS).?°
Aerial applicator personnel and others who may
be exposed to OPs tend to use the peripheral
signs—salivation, weakness or tremor. visual
blurring, ete.?? ?°—as a danger warning, and will
use or seek treatment with atropine and/or
pralidoxime to relieve such peripheral symp-
tomatology. Two assumptions underly this
course of action: (1) that OPs are not likely to
affect CNS functions without concomitant pe-
ripheral signs, and (2) that drugs such as atro-
pine and pralidoxime which will eliminate
peripheral symptoms will a/so restore CNS func-
tions to normal. If either assumption is wrong,
there is a substantial and unrecognized hazard
present to aerial applicator personnel in the form
of unrecognized CNS dysfunctions. There is
little direct evidence concerning either assump-
tion in the literature. The following experiments
were undertaken to test assumption No. 1, to see
whether an OP, mevinphos, would produce elec-
trophysiological signs of CNS dysfunction at
doses too low to produce peripheral symptoms.

Neurophysiological studies on OPs and other
acetylcholinesterase inhibitors (AChEIs) have
tended to center on their effects on the ascending
reticular activating system (ARAS).71 181
Thus, OP influences on brain function are usually
thought of in terms of minor effects on alertness
which may not involve significant performance
decrements. However, cholinergic linkages are
not unique to the ARAS.*§ 129151721 Hippo-
campus, for example, has a cholinergic in-
put#%°75 The fibers originate in and come

through the septum,‘ ®***° terminate on or near
the hippocampal cell body?+* and are excita-
tory.‘ The powerful excitatory effects of
iontophoretically administered ACh on hippo-
campal cells‘ ** suggest that even small doses of
OPs could induce hippocampal dysfunctions
without necessarily having much effect on other
brain nuclei. Hippocampus is thought to be
necessary for certain alerting, orienting and
short-term memory storage functions.t¢® 1 36 21 2
Thus, a lightly-poisoned pilot required to remem-
ber a new procedure or orient to an emergency
situation might well be unable to do so, even in
the absence of overt signs of poisoning. There-
fore, the following experiments were initiated to
test whether doses of the OP mevinphos (Phos-
drin),, too small to produce peripheral signs or
symptoms, could alter hippocampal electrophys-
iology. These experiments were undertaken
simultaneously with, and in cooperation with the
similarly directed behavioral experiments of
Dr. M. F. Lewis (AAC-116), reported in OAM
Report FAA~AM-72-29,

II. Methods.

Squirrel monkeys with chronically implanted
intracerebral electrodes were the experimental
subjects, At the beginning of this series, careful
consideration was given to the use of human
yolunteers or poisoned aerial applicator person-
nel in experiments studying EEG and visual
evoked potentials recorded from the scalp. The
necessary organophosphate doses were deemed
too hazardous for volunteers while the problem
of evaluating how much of what chemicals the
applicators had absorbed, together with the prac-
tical problems of finding them and getting the
measurements, made the use of aerial applicator
personnel impractical. The major recording
electrodes were an insulated stainless steel wire
electrode pair (0.25 mm diameter and 1.0 mm
tip separation) placed in the right side ventral