Effects of repeated administration of soman on schedule-controlled behavior and brain in the rat

Neurotoxicologyand Teratology, Vol. 12, pp. 47-56. ©Pergamon Press plc, 1990. Printed in the U.S.A.

0892-0362/90 $3.00 + .00

Effects of Repeated Administration of Soman on Schedule-Controlled Behavior and Brain in the Rat NORMAN HYMOWlTZ,* ANDREA PLOSHNICK,t LOIS LAEMLEt AND HENRY BREZENOFF:~

Department of *Psychiatry, y-Anatomy and SPharmacology University of Medicine and Dentistry of New Jersey, New Jersey Medical School 185 South Orange Avenue, Newark, NJ 07103 R e c e i v e d 20 M a r c h 1989

HYMOWITZ, N., A. PLOSHNICK, L. LAEMLE AND H. BREZENOFF. Effects of repeated administration of soman on schedule-controlled behavior and brain in the rat. NEUROTOXICOL TERATOL 12(1) 47-56, 1990. --The effects of repeated SC administration of soman on schedule-controlled performance and brain pathology were studied in the rat. Soman suppressed response rates in both components of a multiple fixed interval 50-sec fixed-ratio 25 (mult. F150-sec FR 25) schedule of reinforcement, although all animals revealed marked tolerance to repeated drug administration. Response rates generally recovered to baseline levels within 1-3 sessions. Three of the six animals studied, however, demonstrated marked deterioration of steady state schedule performance, particularly during the FI 50-sec component of the multiple schedule. Compared to untreated controls, all soman-treated animals exhibited pathological changes in brain. The most salient finding was glial cell proliferation in layer 4 and deep parts of layer 3 of the cerebral cortex. Glial cell proliferation was most marked in animals that exhibited deterioration of steady state schedule performance. Soman Schedule-controlled behavior Hippocampus Glial cells Rats

Fixed-interval schedule

Fixed-ratio schedule

Brain assay

Cortex

abnormal brain pathology than rats which had received soman and were normal in behavioral tests. These findings are consistent with previous work (12) which showed that disruption of operant behavior in rats following soman administration was related to the degree of underlying neuropathology. The issue of whether asymptomatic animals remain free of neuropathology, however, merits more attention since Raffaele et al. (16) sacrificed their animals approximately four months following administration of soman. It is possible that a longer time period is required to detect the development of pathology in otherwise asymptomatic animals. Behavioral studies in our laboratory with subcutaneous (SC) administration of soman (70-90 Ixg/kg) in rats showed that soman suppressed response rates in both components of a multiple fixed interval 50-sec fixed ratio (mult. FI 50-sec FR 25) schedule of food reinforcement (2). At the highest doses, soman caused tremors or mild tonic seizures, with hind limb abduction in some of the animals. The suppression of response rates was correlated with inhibition of AChE in cortex, striatum, hippocampus, hypothalamus, and brainstem. The somatic symptoms and changes in performance may reflect underlying degenerative neuropathology (13), although it is important to more directly study the relationship between changes in behavior following soman and brain pathology. Studies of schedule-controlled performance lend themselves to long-term investigations of drug effects (21), affording opportunity for analyses of changes in behavior resulting from acute and chronic exposure to the drug. The present studies extend the

THE organophosphate acetylcholinesterase (ACHE) inhibitors were developed shortly prior to and during World War II, first as agricultural insecticides and later as potential chemical warfare agents (7). The extreme toxicity of organophosphate compounds poses important challenges to the behavioral and toxicological communities (21). While it is clear that excessive exposure to organophosphate may produce respiratory distress, degenerative neuropathology, and death (4), less is known about the effects of low level exposure. Acute exposure of humans to nonlethal levels of organophosphate inhibitors of acetylcholinesterase (ACHE) may lead to difficulty in concentration, mental confusion, drowsiness, disturbance in information processing and psychomotor speed, memory and language deficits, and emotional stability (8). Humans (5) and monkeys (3) that have been exposed to organophosphate compounds and do not reveal overt signs of toxicity or behavior change still may experience long term modification of electroencephalograms. Moreover, sublethal soman may produce central nervous system lesions which are not seriously incapacitating at the time of exposure, but which may worsen in time, affecting behavior as well as the health of the animal (21). The behavioral effects of a single dose of soman (50 Ixg/kg or 85 I~g/kg, SC) on activity levels in an open field, reactivity to external stimuli, and t-maze performance in rats have been studied (16). Some of the animals showed increases in activity and reactivity and learning deficits in the maze. The performance of other rats did not differ from controls. Rats which received soman and were abnormal in behavioral tests were more likely to have 47

48

HYMOWITZ, PLOSHNICK, LAEMLE AND BREZENOFF

analysis of neuropathology in asymptomatic and symptomatic soman-treated animals. The operant-conditioning paradigm was used to study the behavioral effects of acute administration of soman, recovery of response rates following soman administration, and long-term changes in baseline performance following repeated and chronic administration of sublethal doses of soman. Six animals that did not reveal overt signs of seizure activity in response to soman were selected from a larger pool of animals which served in a study of the behavioral effects of soman on schedule-controlled behavior. The six animals were followed for an extended period of time, and they were sacrificed for pathological examination of brain. METHOD

TABLE

1

SOMAN DOSE FLOW CHART

INITIAL DOSE OF SOMAN

1

[

I

i

BEHAVIOR SUPPRESSED TO 80% OF BASELINE

BEHAVIOR NOT SUPPRESSED TO 80% OF BASELINE

i

REPEATED ADMINISTRATION OF SAME DOSE OF SOMAN

INCREASE SOMAN DOSE

--I

Subjects Six male Sprague-Dawley rats, initially weighing 350-375 g, served in the study. They were maintained at 80% of their free-feeding body weight and housed individually with water freely available.

BEHAVIOR NOT SUPPRESSED TO 80% OF BASELINE

I INCREASE SOMAN DOSE OVER SUCCESSIVE DAYS

Behavioral Studies Behavioral experiments were conducted in single-lever operant conditioning chambers and associated sound-attenuating enclosures (Coulbourn Instruments). Chamber lights served to distinguish between the two components of a multiple schedule. Programming was accomplished with a Digital System PDP-8 Computer, and Noyes food pellets (0.045 g) served as reinforcers. The animals were trained to press a lever under a mult. FI 50-sec FR 25 schedule of food reinforcement. Six 10-min components alternated successively throughout a 60-min session, and the component in which the session was initiated was determined randomly each day. White masking noise signaled the start of the session and remained on throughout the session, The performance of the animals was monitored for 25-30 sessions prior to the initial administration of soman. During this time, each animal acquired stable rates of lever pressing. Some of the animals were exposed to physostigmine salicylate, a "short-acting" AChE inhibitor, and N-(4-diethylamino-2-butynyl)-succinimide(DKJ-21), a centrally acting antimuscarinicagent, as well as soman, during the course of study. At the doses studied, physostigmine and DKJ-21 had little, if any, influence on behavior. Their possible antagonism of soman's effects will be reported elsewhere. On drug days, soman was injected SC immediately prior to placing the rat in the chamber. A minimum of 5 weeks separated each soman administration, during which time the behavior of the animals was studied in the chamber 5 days per week, with limited feeding on weekends. The behavior of the animals was studied repeatedly under the same dose of soman or under an ascending series of doses, depending on whether or not behavior was suppressed to less than 80% of baseline (Table 1). If behavior was consistently suppressed by a given dose of soman, the animal was not exposed to a higher dose. Otherwise, the dose was increased in gradual increments. If the dose was increased and the behavior of the animal still was not affected, the dosing strategy was changed to avoid undesirable toxic effects (seizures and/or death). Instead, soman was administered to the animal on three successive days. The ineffective dose which was given five weeks earlier was readministered on Drug Day 1 and lesser doses were given on Drug Days 2 and 3 (e.g., 75, 40, and 40 txg/kg of soman on Days 1, 2, and 3, respectively). If the performance of the animal was suppressed to 80% of baseline on Drug Day 2, soman was not administered on Drug Day 3. Soman was not administered on more than three successive days no matter what degree of response suppression was observed.

Drugs Soman (pinacolyl methylphosphonofluoridate) was obtained from the U.S. Army Medical Research and Development Command and was maintained at -70°(2. Aliquots were diluted in saline immediately prior to use such that the volume of each injection was 1.0 ml/kg body weight.

Histological Examination Following completion of the behavioral studies, rats were given intraperitoneal injections of phenobarbital sodium (40 mg/ kg), and the brains were fixed by intracardiac perfusion with physiological saline followed by either Bouins solution or 2% paraformaldehyde--4% glutaraldehyde. Brains were removed, embedded in paraffin and sectioned at 10 Ixm in the coronal plane. Selected tissue sections were stained with cresyl violet or with hematoxylin and eosin. Other sections were examined by light microscopic immunocytochemistry for S-100 or glial fibrillary acidic protein (GFAP) using the avidin biotin method. Cytoarchitecture and immunostaining in the soman-treated rats were compared with a control group which had not been injected with soman. For the immunocytochemical procedure, sections were deparaffinized, rehydrated to phosphate buffered saline (PBS, 0.1 M, pH 7.4) and sequentially exposed to 1) normal rabbit serum (0.3 hr); 2) antiserum to GFAP (INCstar, Inc., 1:250, 0.5 hr) or S-100 (INCstar, 1:600, 00.5 hr); 3) biotinylated antibody solution (Vector Labs, 0.5 hr); Vectastain ABC reagent (Vector Labs, 1 hr); and 4) peroxidase substrate solution (0.1% diaminobenzidine tetrahydrochloride, Sigma, in 0.1 M Tris buffer pH 7.2, to which an equal volume of 0.02% hydrogen peroxide is added, 7 min). Sections were rinsed in PBS after steps 2 and 3, and in water after step 4, counterstained with cresyl violet, dehydrated, and coverslipped. Immunoreactive cells were identified by the dark brown reaction product which filled the cell body and cytoplasmic processes. Staining was judged specific by the absence of reaction product when the primary antiserum was replaced by buffer or by normal rabbit serum, and by the completely different patterns of immunoreactivity obtained when the primary antiserum was replaced by antiserum to vasoactive intestinal polypeptide (VIP) or somatostatin (SRIF).

REPEATED ADMINISTRATION OF SOMAN

49

TABLE 2 EXPERIMENTAL TREATMENTS FOR ANIMALS WHICH REVEALED DETERIORATION OF BASELINE AND FOR ANIMALS WHO DID NOT REVEAL DETERIORATION OF BASELINE SCHEDULE-MAINTAINED PERFORMANCE

Rat 214" 200* 137" Mean 58? 74? 1397 Mean

Sessions

Soman Occasions

Total Soman (p,g/kg)

Highest Soman Dose (p,g/kg)

Other Drugs

16 16 15 15.66 9 5 17 10.33

16097 1854 1376 1613 1470 650 1700 1273

155§ 190§ 150§

Yes¶ Yes¶ Yes¶

225§ 170§ 125§

No No Yes¶

339 337 290 252 173 381

*No deterioration of baseline schedule-maintained performance. ?Deterioration of baseline schedule-maintained performance. ~:Occasions on which soman was administered over three successive days are considered as one soman occasion. §Dose given over three successive days. ¶Physostigmine (SC) and/or DKJ-21 (IP).

RESULTS

Behavioral Studies Table 2 summarizes the behavioral treatment of each animal. The animals were studied in the chamber for a large number of sessions (173-381 sessions), resulting in several years of study. Similarly, the animals were exposed to soman on numerous occasions, ranging from 5 times for Rat 74 to 17 times for Rat 139. Other animals studied in our laboratory had longer experimental histories and more exposures to soman without revealing signs of ill health or deterioration of operant performance. The total exposure to soman was quite high (Table 2), and it is likely that the highest dose administered would have proved lethal to a naive animal, even if it was administered over three successive days, as

was the case in the present study [see also (19,20) which revealed marked changes in LDso doses of soman following repeated soman administration]. All of the animals acquired stable baseline response rates (Table 3). Prior to the introduction of soman, response rates during the FI 25-sec schedule of food delivery were lower than during the FR 25 schedule of reinforcement, quarter-life values (the percent of the 50-second interval which elapsed before 25% of the responses were made) ranged from a low of 31 for Rat 74 to a high of 59 for Rat 58, and the animals quickly obtained the food pellet as soon as it was available under the FI 25-sec schedule (Table 3). Examination of computer printouts (not shown) showed characteristic schedule-maintained performance. Behavior maintained by the FR 25 schedule was characterized by rapid response bursts (ratio runs) which ended in the delivery of the food pellet. The ratio run was followed by a postpellet pause. Under the FI 50-sec schedule, characteristic negatively accelerated "scalloped" response rates were maintained, with relatively little responding during the initial portion of the interval. Quarter-life values were quite high for all of the animals, indicating that the highest rate of responding occurred in the later portion of the interval. When first introduced, the effects of soman on schedulecontrolled behavior were similar to those described in previous studies (2). In some cases, soman led to the complete cessation of responding in both components, with virtually no within-session recovery. Response rates returned to baseline levels in both components in 1-3 sessions. In other instances, intermediate degrees of response suppression were produced. The pattern of responding was influenced by the schedule of reinforcement. The FR 25 schedule generated a pattern of responding in which the animal emitted a rapid uninterrupted burst of responding (ratio run), obtained the food pellet, paused (postreinforcement pause), and then emitted the next burst of responses. Soman administration first led to marked increases in the postpellet pause, with little, if any, effect on the ratio run. As response suppression increased, disruption of the ratio run, as well as increases in the postpellet pause, were observed. For the FI 50-sec schedule, the characteristic scalloped pattern of responding was maintained following soman administration. Responses which occurred early in the interval were most readily suppressed by soman. Quarter life values remained at fairly high

TABLE 3 BASELINE PERFORMANCE AT START AND END OF STUDY Start (Presoman):~

End (Postsoman)§

FI Resp./Sec

FI "1/4 Life" (%)

FI Time (sec) to Obtain Pellet

FR Resp./Sec

FI Resp./Sec

FI "1/4 Life" (%)

FI Time (sec) to Obtain Pellet

FR Resp./Sec

214" 200* 137"

0.84 0.81 1.00

52 42 58

0.87 1.07 0.78

1.12 .99 1.64

0.77 1.16 0.93

56 45 59

0.83 0.82 0.55

2.77 1.37 1.46

Mean

0.88

51

0.91

1.25

0.95

53

0.73

1.87

58t 74? 139t

0.82 1.10 1.02

59 31 47

0.90 0.77 0.77

1.79 2.10 1.27

0.33 0.29 0.73

80 84 66

5.70 12.24 6.16

1.33 1.62 1.58

Mean

0.98

46

0.81

1.72

0.45

77

8.03

1.51

Rat

*No deterioration of baseline schedule-maintained performance. tDeterioration of baseline schedule-maintained performance. 5~Mean of 5 sessions which preceded the first administration of soman. §Mean of 5 sessions which preceded the termination of the study.

50

levels. The time required to press the lever and obtain the food pellet when it became available increased markedly. As the degree of suppression increased, responses throughout the interval were suppressed. Response rates in both components recovered to baseline levels within 1-3 sessions following soman administration, and characteristic patterns of FR- and FI-maintained behavior were reestablished for each animal. For Rats 214, 200, and 137, as well as most other rats studied in our laboratory, drug-free baseline response rates remained relatively stable throughout the study (Table 3). For Rats 58, 74, and 139, considerable deterioration, in terms of response pattern and rate, occurred (Table 3) (see below). Throughout the course of the study, none of the animals revealed signs of seizure activity (e.g., salivation, chewing movements, tremors, " k i n d l i n g " effects, mild tonic seizures, hind limb abduction) or ill health (refusal to eat, loss of weight, respiratory distress), nor was it difficult to handle any of the animals due to hyperexcitability or aggressiveness. These signs of soman toxicity have been observed in other animals following exposure to soman. During sessions in which soman suppressed response rates, the animals remained quiet and calm in the chamber, often lying by the food cup or lever. On some occasions, they appeared heavily sedated. On others they remained responsive to touch, and moved around when prodded or handled. When the session was over and the animals were returned to their home cage, many animals picked up the rat chow and usually began eating as soon as the chow was introduced. Following the initial exposure to soman, the behavior of the animals became quite resistant to the suppressive effects of soman, and variability to a given dose of soman was the rule rather than the exception. Tables 4 and 5 show the responses of Rats 214 and 58, respectively, to soman. These data are representative of the response of the other animals. Prior to the first administration of soman, Rat 214 displayed characteristic schedule-controlled behavior in both components of the multiple schedule. On the day before soman was first introduced, Rat 214 emitted 0.87 responses per sec under the FI 50-sec schedule and 1.22 responses per second under FR 25. The FI quarter-life value was 54%, indicating that the vast majority of the responses occurred in the latter portion of the 50-sec interval. An average of 0.41 sec elapsed between the time a food pellet became available under the FI schedule and the time the rat pressed the lever and obtained the pellet (Table 4). When soman was first introduced (80 ixg/kg), response rates in both components were completely suppressed, with no withinsession recovery. Partial recovery was observed on the day following soman, with a modest reduction of response rates in both components and a slight increase in the time to obtain the food pellet under the FI 50-sec schedule. By the following session, response recovery was virtually complete. These findings for Rat 214 were successfully replicated five weeks later when soman (75 p~g/kg) was readministered (Table 4). Response rates were completely suppressed by soman, considerable response recovery was observed the next day, and baseline response rates and patterns of responding were restored on the following session. The next time soman (75 txg/kg) was administered (4 weeks later), no suppressive effects on schedule-controlled behavior were observed. When soman was once more readministered five weeks later, the behavior of the animal still was insensitive to soman (75 Ixg/kg) (see Table 4, fourth soman occasion). Virtually all of the animals studied in our laboratory, under similar conditions, revealed this kind of variability and growing insensitivity to soman. Table 4 a/so shows for Rat 214 an example of the behavioral response to a much larger dose of soman administered over three

HYMOWITZ, PLOSHNICK, LAEMLE AND BREZENOFF

TABLE 4 REPRESENTATIVE DATA FOR RAT 214" DURING BASELINE AND SOMAN

Soman Occasion

FI Time (sec) to Obtain Food FR Pellet Resp./Sec

FI Resp./Sec

FI "~/4Life"

BLt SOM. (80 p.g/kg) BL$ BL

0.87 0 0.57 1.02

54

0.41

48 45

1.63 0~70

BL SOM. (75 p.g/kg) BL BL

1.00 0.02 0.52 1.00

47

0.52

55 50

2~55 0.49

1.31 0.22 1.00 1.26

BL SOM. (75 Ixg/kg) BL BL

0.87 0.87 0.98 0.76

53 52 54 60

0.39 0.68 0.24 0.50

1.47 1.43 ! .79 1.73

12. BL SOM. (75 Ixg/kg) SOM. (40 p,g/kg) SOM. (40 p.g/kg) BL BL

1.77 2.10 1.53 0.89 1.36 1.87

47 43 52 48 51 45

0.23 0.37 0.42 5.87 0.40 0.28

4.21 4.01 3.09 0.98 3.36 4.02

1.

1.22 0.22 0.73 1.37

2.

4.

*Baseline schedule performance did not deteriorate during the study. tSession immediately preceding administration of soman. $Session immediately following administration of soman. successive days (Table 4, 12th soman occasion). By the twelfth soman occasion, considerable " d r i f t i n g " in the baseline performance of this animal occurred, leading to gradual and marked increases in response rates in both components. Subsequently, there was a gradual decline in response rates, more closely matching earlier response rates. The pattern of responding, however, remained quite stable, with relatively high quarter-life values and a short latency to obtain the food pellet when it became available under the FI 50-sec schedule (Table 4). Again, the 75 txg/kg dose of soman had no obvious effects on behavior. Similarly, administration of 40 Ixg/kg soman on the next day failed to suppress or alter response rates (Table 4). When soman (40 txg/kg) was administered on the third successive day, yielding a total cumulative dose of 155 ixg/kg, intermediate degrees of response suppression were observed in both components. During FI 50-sec food delivery, response rates decreased to 0.89 responses/sec, the time to obtain the food pellet when it became available increased to 5.87 sec, and quarter-life values remained relatively unchanged, suggesting that the pattern of FI responding was maintained despite the lowering of response rates. Fixed-ratio 25-maintained behavior also was suppressed, due to a marked increase in the postpellet pause and disruption of the ratio run, as determined by inspection of computer printouts. As shown in Table 4, response rates increased to predrng levels within two sessions. Table 5 shows a similar profile for Rat 74. While the details may differ, the overall picture is the same as for Rat 214 and the

REPEATED ADMINISTRATION OF SOMAN

51

TABLE 5

TABLE 6

REPRESENTATIVE DATA FOR RAT 58* DURING BASELINE AND SOMAN

EFFECTS OF HIGHEST DOSE OF SOMAN ON RESPONSE RATES IN EACH COMPONENT OF MULTIPLE SCHEDULE

Soman Occasion

FI Resp./Sec

FI Time (Sec) to FI Obtain Food "1/4Life" Pellet

FR Resp./Sec

2.

BLt SOM (80 kl,g/kg) BL

0.89 0.77 0.95

59 65 65

0.89 0.26 0.39

1.94 2.01 1.83

BL SOM (85 p,g/kg) SOM (40 ~,g/kg) SOM (40 iJ,g/kg) BL:~

0.85 0.86 0.91 0 1.13

74 68 65

0.38 0.25 0.60

66

0.56

2.11 2.16 2.02 0 2.01

BL SOM (85 Ixg/kg) SOM (40 Ixg/kg) SOM (40 I~g/kg) BL

0.81 0.61 0.72 0.77 0.59

71 71 73 74 77

0.27 1.16 0.56 0.26 0.93

2.07 1.90 2.06 2.10 1.89

BL SOM (85 IJ,g/kg) SOM (60 o.g/kg) BL BL

0.95 0.71 0.08 0.60 0.72

71 67 56 71 68

0.83 1.51 6.85 0.77 0.91

2.10 1.80 0.64 1.78 1.93

BL SOM (85 p,g/kg) SOM (60 p,g/kg) SOM(60 ~g/kg) BL BL BL

1.07 0.72 0.74 0.70 0.46 0.90 0.81

69 77 75 78 67 71 74

0.27 0.77 0.37 0.34 10.90 0.90 0.51

2.21 2.16 2.08 2.01 2.04 2.16 2.12

3.

4.

6.

7.

*Baseline schedule performance deteriorated during the course of the study. tSession immediately preceding administration of soman. :~Session immediately following administration of soman.

other animals studied in this (Table 6) and other experiments in our laboratory. Initially, 75 and 80 ~g/kg soman had little, or no, disruptive effects on responding. When a higher dose of soman was administered over three successive days (Table 5, 3rd soman occasion), marked response suppression was observed by Day 3, followed by full response recovery by the next session. Five weeks later, the same 165 I~g/kg dose of soman administered over three successive days had virtually no effect on schedule performance (Table 5, 4th soman occasion). When the dose was increased (Table 5, 6th soman occasion), marked response suppression was observed on the second day of soman administration. Response rates in both components were suppressed, the time to obtain a food pellet under FI 50-sec food delivery increased and quarter-life values remained intact. Response recovery was virtually complete within two sessions. When soman was readministered five weeks later (Table 5, 7th soman occasion), the behavior of the animal was highly resistant to the suppressive effects of soman, although some residual effects of soman were observed on the first

Rat 214t 200t 137t 58:~ 74:~ 139:~

No. of Time Response Rates Reduced to Below 80% of BL Per Occasion

Mean %

BL*

Soman (i.Lg/kg)

FI

FR

FI

FR

155 190 150 225 170 125

12 88 57 44 0 23

34 102 52 46 0 42

2/2 1/3 3/3 1/2 1/1 1/2

2/2 0/3 2/3 1/2 1/1 2/2

9/13

8/13

*Mean of three drug-free baseline (BL) sessions which preceded administration of soman. tNo deterioration of baseline schedule-maintained performance. :~Deterioration of baseline schedule-maintained performance.

postsoman session in the component of the multiple schedule associated with the FI 50-sec schedule of food delivery (e.g., low response rate and increased latency to obtain food pellet when it became available). Table 3 shows mean response rates and "quarter-life" values for each animal for 5 sessions preceding the first administration of soman (Session 20-25) and for five sessions preceding the termination of the study. Despite some variability and " d r i f t i n g , " each animal sustained relatively high rates of responding under the FR-25 schedule during the entire course of the study. H-maintained behavior, however, seemed to be adversely affected in Rats 58, 74, and 139 and relatively unaffected in Rats 214, 200, 137, despite the fact that none of the animals revealed signs of seizure activity or ill health and all of the animals were exposed to comparable amounts of soman (Tables 2 and 6). The deterioration of FI 50-sec schedule performance is reflected in a marked lowering of FI 50-sec response rates in Rats 58, 74, and 139, a marked increase in quarter-life values for Rats 58 and 74, and a marked increase in the time between the availability of the food pellet and the response to produce it (Rats 58, 74, 139) (Table 3).

TABLE 7 GLIAL CELLS (REPRESENTED AS NUMBER OF GLIAL CELLS PER 100 CORTICAL NEURONS) IN LAYER 4 OF THE MOTOR CORTEX

Animal R-54 R-56 S-74 S-58 S-214 S-200

Treatment and Condition Normal, untreated control Normal, untreated control Soman-treated, severely deteriorated Soman-treated, severely deteriorated Soman-treated, asymptomatic Soman-treated, asymptomatic

Glial Cells Per 100 Neurons 10 8 47 59 19 20

*Number of glial cells represents the average of 10 to 20 counts per animal.

52

HYMOWITZ, PLOSHNICK, LAEMLE AND BREZENOFF

,ira

,¢

i

II

%,

J

%

f

! 0p

i 41

11.

2

3

4

S .

FIG. 1. Photomicrographs through layers 1 to 5, as indicated in fight margin, of the motor cortex of soman-treated rats. Sections have been stained with GFAP, a histochemical marker for astrocytes, and counterstained with cmsyl violet for cytoarchitecture. (A) Rat 214, an animal whose baseline behavior did not deteriorate. Glial cell nuclei are indicated by arrows. Note the absence of staining with GFAP (for comparison see Fig. 2B). (B) Rat 58, an animal whose baseline behavior did deteriorate. Note the increase in glial cell density in layer 4, and the absence of staining with GFAP. Compare glial cell density in Fig. 3A and B with Fig. 2A, which shows layer 4 of a control rat.

Normally, rats obtain the food pellet as soon as it becomes available. The marked delay in obtaining the food pellet following soman in some of the animals seemed to be the most sensitive and salient indication that baseline performance was adversely affected by soman. The steady state performance of Rats 214,200, and 137 did not reveal increases in the latency between pellet availability and lever pressing, although some " d r i f t i n g " of response rates was observed in each of the animals during the course of the study. However, the pattern of responding in each component of the multiple schedule remained intact. The baseline performance of these animals did not reveal any signs of deterioration throughout the course of the study, although they received as much soman, or

more, as animals which experienced changes in baseline performance. It is of interest to examine brains of animals who experienced such deterioration and those that did not. While all of the animals were asymptomatic from the point of view of their physical health, it is possible that the deterioration of schedule performance in Rats 58, 74, and 139 was associated with underlying neuropathology.

Brain Assay Cytoarchitecture and immunostaining in the three somantreated rats (58, 74, 139) whose baseline behavior deteriorated during the study and three soman-treated rats (200, 214, 137) whose behavior did not deteriorate were compared with a control

REPEATED ADMINISTRATION OF SOMAN

53

FIG. 2. (A) Layer 4 and superficial region of layer 5 of the motor cortex of a control rat, which has not been exposed to soman. Note small number of glial cell nuclei and absence of GFAP staining. As in Fig. 1, tissue sections have been stained for GFAP to demonstrate astrocytes, and counterstained with cresyl violet. Magnification: x 350. (B) White matter (WM) and deep border of layer 6 of same tissue section shown in (A). Note prolific staining of astrocytes. Magnification: × 400. group which had not been injected with soman. Rats 214, 200, 137, and 139 were sacrificed within one month of their last exposure to soman. Rats 58 and 74 were followed for 4 and 5 months, respectively, without additional exposure to soman once they revealed consistent signs of behavioral deterioration. Hence, a minimum of 4 and 5 months separated their last exposure to soman and sacrifice. Analyses focused on the motor cortex and the hippocampus, two areas reported to undergo pathological changes following single, near lethal doses of soman (13-15). Cortex. Histological examination of all soman-treated animals revealed evidence of neuropathology. Neuronal alterations in animals that failed to reveal signs of behavioral deterioration following soman were not as extensive as those in animals in which schedule-controlled performance had deteriorated. In all cases, pathological changes were more subtle than those reported following single large doses of soman (13-15). Compared with the control group, the motor cortex of all soman-treated animals was characterized by a marked increase in the number of small nuclei which appeared to belong to glial cells. This reaction was most prominent in layer 4 and the deeper portions of layer 3. Stained tissue sections viewed by light microscopy were simultaneously projected by a Dage TV camera mounted on the microscope onto a Panasonic video monitor at a magnification of 1,000 × . The projected microscopic field was analyzed using an IBM PC and a Southern Micro Instruments image analysis system. The number of glial cells per 100 cortical neurons were recorded (Table 7). Glial cells in soman-treated rats whose baseline behavior had not deteriorated (Fig. 1A) were 1.6 times as numerous as in control rats (Fig. 2A). In animals whose behavior had deteriorated there was a 5-fold increase in glial cells over control rats (Figs. 1B, 2A). In order to more accurately determine the nature of the glial cell reaction, sections from behaviorally deteriorated, nondeteriorated,

and control rats were stained for immunoreactivity to GFAP and to S-100, two histochemical markers for astrocytes (Fig. 2B). The identical pattern of immunoreactivity obtained (many cells stained in the white matter and below the pia mater, but few to none in layers 3 and 4) suggested that these were not astrocytes. Rather, the amount of cytoplasm and staining characteristics of the additional glial cells were similar to those described for medium shade oligodendrocytes (I0). Immunocytochemical staining also revealed a subtle increase in astrocytes in layer 5 of soman-treated rats as compared with control rats. Hippocampus. In the hippocampal region, only one of the most severely deteriorated rats (Rat 58) showed evidence of neuronal degeneration. This occurred in the dentate gyms, along the border of the granule cell layer. Necrotic neurons appeared as angular to fusiform cells with intensely staining cytoplasm, and nuclei which were eccentrically located, decreased in diameter, and sometimes invaginated (Fig. 3B). In contrast, normal granule cells which comprise this layer are small, round, and regularly arranged, with prominent round nuclei (Fig. 3A). DISCUSSION The present study showed that it is possible to study the acute and chronic behavioral effects of repeated administration of soman in individual animals without compromising their health or vitality. In general, soman suppressed schedule-controlled behavior in a dose-dependent manner. Initially, soman suppressed response rates in both components of the mult. FI 50-sec FR 25 schedule of reinforcement. Response rates recovered to baseline levels within 1-3 sessions. This finding replicates previous reports (2) and documents the sensitivity of schedule-controlled behavior to acute effects of soman. However, when soman was repeatedly administered to the same animal, marked variability in response to soman emerged. A dose of soman which previously suppressed

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FIG. 3. Dentate gyrus of two soman-treated rats, stained for GFAP and cresyl violet. (A) Rat 200, an animal whose behavior did not deteriorate during the course of the study, shows normal cytoarchitecture of this area. The granule cell layer is homogeneous, and comprised of cells with uniform small, round nuclei. Arrows = astrocytes stained for GFAP. Magnification: x 400. (B) Rat 58, an animal with severe behavioral deterioration. Note the decreased thickness of the granule cell layer, and its heterogeneous cytoarchitecture. Large arrows = necrotic neurons; small arrows = astmcytes stained for GFAP. Magnification: x 400.

response rates often no longer suppressed behavior. Similar tolerance to repeated administration o f subsymptomatic doses o f soman has also been reported by Russell et al. (18). Previous studies with intraperitoneal administration o f soman (6) showed that it was necessary to separate soman administration by 3-5 weeks to avoid tolerance to s o m a n ' s suppressive effects.

The same time period was required for A C h E levels in gut to recover to baseline levels (1). Unpublished studies in our laboratory showed that 3-5 weeks also were necessary for A C h E levels in brain to recover to baseline levels following soman (SC) administration. The animals in the present study revealed marked tolerance to the suppressive effects o f soman despite the fact that

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a minimum of five weeks separated soman administration. It is possible that more time between administrations of soman is necessary for studies of the behavioral effects of repeated administration (SC) of soman within individual animals. Although massive amounts of soman ultimately were administered to each animal, none of the animals developed seizure activity or signs of ill health. It is unlikely that the administration of other drugs during the course of the study offered any protection from the disruptive effects of soman on baseline performance. Numerous other animals who were not exposed to the other compounds and who received repeated administration of soman also did not reveal changes in baseline performance or signs of soman toxicity. Moreover, the other drugs which were administered failed to antagonize the suppressive effects of acute soman administration. Three of the six animals, however, revealed clear evidence of behavioral deterioration, particularly under the FI 50-sec schedule of food delivery. While FR 25 food-maintained behavior continued at a relatively high rate, the response rate during the FI 50-sec schedule slowed markedly, quarter-life values increased, and the time to obtain a food pellet, once it became available, increased. Presumably, the deterioration of baseline performance reflected underlying neuropathology, since the health and vitality of the animals did not appear compromised. Analyses of brain tissue supports this conclusion. The current study showed that chronic exposure to sublethal doses of soman causes pathological changes in the CNS. Cellular reactions were more subtle than in organophosphate poisoning which results from near-lethal doses 0 3 - 1 5 ) . In our animals, the most prominent pathological change was glial cell proliferation in layer 4 and deep parts of layer 3 of the cerebral cortex. The severity of the baseline behavioral deterioration corresponded to the degree of pathology observed. However, glial proliferation was evident in all soman-treated animals. Glial cell populations in the cerebral cortex include astrocytes, microglia, and oligodendrocytes. Ling et al. (10) further classified oligodendrocytes in the rat cerebral cortex as light, medium, and dark. Our data suggest that the cells of interest represent a specific subpopulation of oligodendrocytes. Their failure to stain with either S-100 or GFAP indicates that they are not astrocytes. The amount of cytoplasm and nuclear staining characteristics are compatible with descrip-

tions of medium shade oligodendrocytes. Finally, the numbers of these cells counted in control animals (less than ten per one hundred cortical neurons) correspond closely to those reported by Ling and Leblond (9) for medium oligodendrocytes. Proliferation of oligodendrocytes has been reported following various kinds of brain insults (11,17). Comparing our data with a previous report by McLeod, Singer, and Harrington (13), we believe that areas which are characterized primarily by glial proliferation in our study are those which underwent marked cell death following near-lethal doses of soman in the former study. In that study, rats which experienced convulsions following large doses of soman (a single injection of 165 Ixg/kg) subsequently developed lesions in the neocortex and hippocampus. Two weeks after injection, the lesions were characterized by marked glial cell proliferation. The authors attributed these lesions to the convulsions and resulting hypoxia. In our study, glial cell proliferation in the same area occurred in the absence of convulsions or hypoxia. Petras (15), in a study of the effects of soman in cat and rat, reported axonal degeneration in a cat that remained asymptomatic after two intermuscular injections of soman administered one week apart. In the rats, pathological changes were not observed in animals which remained asymptomatic, and in those that developed overt neurological symptoms, the pattem of degeneration did not resemble that caused by experimental fetal hypoxia. In our study, reactions in the hippocampus, as in the motor cortex, were less severe than those reported following near-lethal doses of soman (15). The reaction seen in our tissue may represent early stages in the severe cell loss observed following near-lethal doses. The observation that all soman-treated animals responded with glial proliferation in the motor cortex, while definitive changes in the hippocampus could be seen in only one animal, raises the possibility that the hippocampus may be more resistant to the cumulative effects of low doses of soman than the neocortex. ACKNOWLEDGEMENTS This work was supported by USAMRDC contract DAMD17-82C-2172. The authors would like to thank John McGee and Karel Campbell for their assistance in conducting the studies.

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