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Effects of trialkyltins on the schedule-controlled behavior of the pigeon
Neurotoxicologyand Teratology, Vol. 9, pp. 67-74. Copyright©PergamonJournals Ltd., 1987. Printed in the U.S.A.
0892-0362/87$3.00 + .00
Effects of Trialkyltins on the Schedule-Controlled Behavior of the Pigeon D. E. M c M I L L A N , * G. R. W E N G E R , * M. J. B R O C C O , .1 S. O. I D E M U D I A * A N D L. W. C H A N G * t
*Department of Pharmacology and Interdisciplinary Toxicology and tDepartment of Pathology University of Arkansas for Medical Sciences, 4301 W. Markham Street, Little Rock, AR 72205 R e c e i v e d 11 F e b r u a r y 1985 McMILLAN, D. E., G. R. WENGER, M. J. BROCCO, S. O. 1DEMUDIA AND L. W. CHANG. Effects oftrialkyltins on the schedule-controlled behavior of the pigeon. NEUROTOXICOL TERATOL 9(1) 67-74, 1987.--Male White Carneaux pigeons trained to respond for food under a multiple fixed-ratio fixed-interval schedule of reinforcement were given single injections of trimethyltin (TMT), or triethyltin (TET). A dose of 0.3 mg/kg TMT produced no effect on behavior, while a 1.0 mg/kg dose was a threshold dose and 1.75 mg/kg produced behavioral changes that persisted for months in some birds. TMT produced effects on responding under the multiple schedule at approximately the same doses that produce neuronal damage in the hippocampus and the brain stem of the pigeon. Higher doses given to untrained birds produced signs of extensive neurological damage. A dose of 1.0 mg/kg of TET decreased rates of responding under both schedule components three hours after administration, but behavior usually had recovered by the next day. Doses of 3.0 and 5.6 mg/kg had similar effects, but responding did not recover for several days. Some birds showed significant rate increases, especially under the fixed-interval component several days to several weeks after TET administration. Doses greater than 10 mg/kg TET were lethal. Dose-effect curves for the effects of d-amphetamine, chlorpromazine and morphine on responding under the multiple schedule were determined for some birds before and one month after 1.0 and 1.5 mg/kg of TMT. TMT shifted the dose-effect curve for d-amphetamine to the right, but it did not produce systematic changes in the dose-effect curves for morphine and chlorpromazine. Trialkyltins
Pigeons
Schedule-controlled behavior
METHOD
BECAUSE organic tin compounds have been used widely in industry and because they produce specific localized lesions in the brain [1, 2, 12], there has been considerable interest in the behavioral toxicology of these compounds [1, 6, 10, 13, 16, 20, 21]. Although operant behavior has been used to describe the effects of many chemicals on behavior [1 I], only a few studies have reported on the effects of alkyltins on schedule-controlled behavior [17,21]. The present study provides a comparison of the effects of trimethyltin (TMT) and triethyltin (TET) on responding maintained by a multiple schedule of food presentation in the pigeon. The pigeon was chosen because there is a large literature on the effects of drugs and toxic chemicals on schedule-controlled behavior in pigeons and because birds have been suggested as subjects in several guidelines for neurotoxicity testing [14,18]. Pharmacological challenge has been shown to be useful for evaluating exposure to many types of toxicants [3, 4, 12] and in some instances has proven exceedingly sensitive in detecting behavioral effects [9]. Therefore, the effects of d-amphetamine, chlorpromazine and morphine were determined before and after TMT administration.
Animals A total of 21 adult male White Carneaux pigeons (Palmetto Pigeon Plant, Sumter, SC), used to measure the effects of alkyltins on schedule-controlled behavior, were maintained at 80% of their free-feeding weights throughout all experiments. Their 80% body weights ranged from 410 to 518 g. Birds were fed mixed grain (Ralston-Purina pigeon diet). Crushed oyster shell was freely available in home cages.
Materials and Dosing TMT hydrochloride solutions were prepared for administration as 0.3, 1.0 and 1.75 mg/ml and TET sulfate solutions as 1.0, 3.0 and 5.6 mg/ml. Doses were calculated as the salts. Distilled water was given as a control injection and distilled water served as the solvent for both alkyltin salts. Injection volumes were 0.1 ml/100 g of body weight. All injections were given 3 hours before behavioral testing by the intramuscular route.
1Present address: Institut de Recherche Servier, 14 Due du Val d'Or, Suresnes, 92150, France.
67
M c M I L L A N ET AL.
68 03
t.O MG/KG T R I M E T H Y L T I N
MG/KG TRIMETHYLTIN
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2 3 FIXED INTERVAL
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125 l
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RATIO e------=
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FIXED RATIO
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FIG. 1. Effects of 0.3 mg/kg TMT on responding under the mult schedule. Each point represents the rate for a bird during a single test session, beginning 3 hr after TMT administration on day 1. Filled points fall more than two standard deviations from the mean. Bird 126 (©): Mean FR rate=2.30 responses/sec. Mean FI rate=0.33 responses/sec. Bird 127 (A): Mean FR rate=2.05 responses/sec. Mean FI rate=0.68 responses/sec. Bird 128 (El): Mean FR rate=2.53 responses/sec. Mean FI rate=0.19 responses/sec.
In drug interaction experiments, d-amphetamine sulfate, chlorpromazine hydrochloride and morphine sulfate were given intramuscularly 10 min before the session. Two birds received an ascending dosage series and one a descending dosage series. After determination of the dose-effect curves with two observations at each dose level in each of 3 birds, 1.0 mg/kg TMT was given and behavioral testing was continued for one month, after which the dose-effect curve was redetermined for each of the 3 drugs. After redetermination of the dose-effect curves, a. 1.5 mg/kg dose of TMT was given and after an additional month of behavioral testing the dose-effect curves for the 3 drugs were redetermined again.
Apparatus for Behavioral Testing Behavioral testing was conducted in two sound- and light-attenuating chambers similar to those described by Ferster and Skinner [5]. The chamber was illuminated by a 28 V DC light and contained a translucent plastic response key (2.0 cm in diameter) which was centrally mounted in the chamber wall 22 cm above a wire-mesh floor. The response key could be transilluminated by red or blue lights. The minimum force required to operate the key was about 0. I N. Centered below the key and 6 cm above the floor was a rectangular opening through which the pigeon could be given access to mixed grain. During access to grain all lights except those illuminating the feeder were off. Relay programming and recording apparatus housed in the adjacent room controlled the delivery of grain and recorded the pattern of key pecking. A masking noise was supplied by a speaker mounted inside the chamber.
Procedure Pigeons performed Monday through Friday under a multiple fixed-ratio, fixed-interval schedule (mult FR FI) of food
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FIG. 2. Effects of 1.0 mg/kg TMT on responding under the mult schedule. Each point represents the rate for a bird during a single test session, beginning 3 hr after TMT administration on day 1. Filled points fall more than two standard deviations from the mean. Bird 1 (©): Mean FR rate= 1.56 responses/sec. Mean FI rate=0.43 responses/sec. Bird 4 (A): Mean FR rate=5.04 response/sec. Mean FI rate=l.28 responses/sec. Bird 101 ([]): Mean FR rate=2.65 responses/sec. Mean F1 rate=0.95 responses/sec. Bird 102 (~): Mean FR rate= 1.68 response/sec. Mean F1 rate=0.58 response/sec. Bird 106 (V): Mean FR rate=2.20 responses/sec. Mean FI rate=0.46 responses/sec. Birds 1 and 4 received additional injections of TMT beginning in the third week so their data are shown for only the first two weeks after TMT administration.
presentation. In the presence of a blue light, the 30th key peck (FR 30) produced 4-sec access to grain. In the presence of a red light, the first response after 5 min (F! 5) produced 4-sec access to grain. The FR and FI components alternated after each food presentation. If a bird failed to respond within 30 sec after 5 min had elapsed during the FI component, the schedule changed to the FR component. If the bird failed to make 30 responses within 40 sec during the FR component, the schedule changed to the F1 component. Sessions were started in the FR component I0 rain after the pigeon was placed in the chamber and were terminated after 6 presentations of each schedule component. Upon termination of the behavioral experiments the pigeons were sacrificed for examination by light microscopy. At sacrifice, pigeons were anesthetized with pentobarbital and were perfused intracardially with saline solution followed with 2.5% buffered glutaraldehyde. Animals were then decapitated and the brains removed carefully, further fixed in Bovins-formalin fixative, and prepared for histopathological examination.
Data Analysis For pigeons responding under the multiple schedule, the rates of FR and FI responding were calculated daily. The quarter life (a measure of the pattern of FI responding) was determined according to the method of Gollub [8]. Data from at least 8 control sessions, before some of which saline injections were given, were used to determine control means and standard deviations. Points falling more than two standard deviations from the control mean after TMT or TET
TRIALKYLTINS
AND PIGEON BEHAVIOR
69
TABLE 1
1.75 MG/KG TRIMETHYLTIN FIXED RATIO
EFFECTS OF TMT AND T E T ON Q U A R T E R L I F E (QL EXPRESSED AS PERCENT)
,,ours Af, er Alkyltin Drug
Dose (mg/kg)
TMT
1.0
TMT
TET
TET
1.75
3.0
5.6
Bird
Control QL (mean _+ 2 SDs)
1 4 101 102 106
41 41 36 31 37
_+ 73 _+ 69 _+ 66 _+ 59 _+ 73
3 53 52 87
40 40 49 54
+ 72 _+ 67 _+ 75 _+ 70
3
27
51
74
45 41
43 53
55 49
43 47
44
49
47
37
X
X
x4,
49
46
40
56
X
X
44
x x
29 36
485 51+
0205 .
134 136 138
27 + 57 36 + 67 32 _+ 50
x
48
48
50
x
29~
37
47
x
33
51 i'
51'~
135 137 150
38 _+ 68 28 _+ 58 42 _+ 52
x x x
39 39 185
42 33 395
42 33 35,~
x: Indicates too few responses occurred to determine a reliable quarter life. J,=Decreased quarter life (more than 2 SDs below the control mean). 1'=Increased quarter life (more than 2 SDs above the control mean).
a d m i n i s t r a t i o n for individual birds were c o n s i d e r e d to be significant. In the drug i n t e r a c t i o n s t u d i e s , g r o u p m e a n s a n d s t a n d a r d d e v i a t i o n s w e r e c a l c u l a t e d for c o n t r o l sessions. T h e effects o f d - a m p h e t a m i n e , c h l o r p r o m a z i n e a n d m o r p h i n e w e r e considered to be significantly different f r o m the c o n t r o l rate w h e n t h e m e a n rate o f r e s p o n d i n g a f t e r a dose was m o r e t h a n 2 s t a n d a r d d e v i a t i o n s f r o m the c o n t r o l m e a n . W h e n d o s e - e f f e c t c u r v e s w e r e c o m p a r e d for d r u g s g i v e n before a n d a f t e r T M T a d m i n i s t r a t i o n , t h e d o s e - e f f e c t c u r v e for e a c h drug was d e t e r m i n e d t w i c e a n d the range was plotted to s h o w t h a t the d o s e - e f f e c t c u r v e was n o t variable. T h e d o s e - e f f e c t c u r v e s for e a c h d r u g a f t e r T M T w e r e c o m p a r e d w i t h the m e a n d o s e - e f f e c t c u r v e b e f o r e T M T with the o b s e r v a t i o n s at e a c h d o s e paired (before a n d after) for a n a l y s i s o f t h e d o s e - e f f e c t c u r v e d i f f e r e n c e s b y the signed r a n k s test [7]. RESULTS
Visual Observation o f Behavioral Effects A t a d o s e of 1.0 mg/kg T M T , no g r o s s b e h a v i o r a l effects w e r e o b s e r v e d . A d o s e o f 1.75 m g / k g p r o d u c e d d e c r e a s e d r e a c t i o n to h a n d l i n g a n d ataxia in 3 o f 4 b i r d s w i t h r e c o v e r y after about 3 days; however, one of these birds showed a d e c r e a s e d food i n t a k e a n d p o s t u r a l c h a n g e s w h i c h lasted for several m o n t h s . A single 3 mg/kg d o s e o f T M T p r o d u c e d t r e m o r s , a t a x i a a n d d e c r e a s e d r e a c t i o n to h a n d l i n g w i t h i n a few h o u r s time. T h e s e birds w e r e sacrificed 24 h o u r s a f t e r T M T a d m i n i s t r a t i o n . Birds g i v e n d o s e s a b o v e 3.0 mg/kg T M T died w i t h i n 24 hr. A t a 1.0 mg/kg d o s e o f T E T , no g r o s s b e h a v i o r a l effects
.
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.
5 FIXED
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6
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7
8
~INTERVAL
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WEEKS
FIG. 3. Effects of 1.75 mg/kg TMT on responding under the mult schedule. Each point represents the rate for a bird during a single test session, beginning 3 hr after TMT administration on day 1. Filled points fall more than two standard deviations from the mean. Bird 3 ((3): Mean FR rate=0.65 responses/sec. Mean F1 rate=0.33 responses/sec. Bird 52 (A): Mean FR rate=l.26 responses/sec. Mean FI rate=0.23 responses/sec. Bird 53 (IS]): Mean FR rate= 1.38 responses/sec. Mean F1 rate=0.14 responses/sec. Bird 87 (O): Mean FR rate=l.95 responses/sec. Mean FI rate=0.34 responses/sec. Bird 53 was sacrificed for histological examination at the end of the 7th week after TMT administration.
w e r e o b s e r v e d . T h e 3.0 mg/kg d o s e p r o d u c e d t r e m o r s , a t a x i a a n d m u s c l e rigidity, but by 24 h o u r s a f t e r the injection, t h e birds no l o n g e r s h o w e d a n y g r o s s s y m p t o m s . T h e 5.6 mg/kg d o s e c a u s e d the birds to collapse w i t h a high d e g r e e o f muscle rigidity. T h e r e w a s a rapid o n s e t ( a b o u t l0 min) o f t r e m ors a n d the birds w e r e u n a b l e to walk or fly. T h e b i r d s app e a r e d to b e n o r m a l at 24 h o u r s , e x c e p t t h a t o n e bird s h o w e d a r e d u c e d food intake at this time.
Effects on Mult FI FR Performance T h e effects of 0.3 mg/kg T M T o n r e s p o n d i n g u n d e r the mult F R FI s c h e d u l e are s h o w n in Fig. 1 for 3 individual birds. A l t h o u g h r a t e s o f r e s p o n d i n g o c c a s i o n a l l y fell m o r e t h a n 2 s t a n d a r d d e v i a t i o n s f r o m the m e a n c o n t r o l , this occ u r r e d only 7 t i m e s in 120 o b s e r v a t i o n s d u r i n g a 4 - w e e k period, indicating t h a t 0.3 mg/kg T M T p r o d u c e d n o signific a n t effect o n r a t e s o f r e s p o n d i n g u n d e r the multiple s c h e d u l e . T h e 0.3 mg/kg d o s e o f T M T did n o t affect the quarter-life values for FI r e s p o n d i n g . Figure 2 s h o w s t h e effects o f 1.0 mg/kg T M T o n rates of r e s p o n d i n g u n d e r the multiple s c h e d u l e o f food p r e s e n t a t i o n . B o t h i n c r e a s e s a n d d e c r e a s e s in rate o f r e s p o n d i n g were obs e r v e d a l t h o u g h the effects were small a n d were n o t obs e r v e d e v e r y day. T h e s e small effects o n r a t e s o f r e s p o n d i n g were o b s e r v e d d u r i n g s o m e s e s s i o n s for t h e e n t i r e 8-week period d u r i n g w h i c h o b s e r v a t i o n s were m a d e . T h e 1.0 mg/kg d o s e of T M T did not affect the quarter-life values for FI r e s p o n d i n g (Table 1). Figure 3 s h o w s t h e effects o f 1.75 mg/kg T M T o n r a t e s of r e s p o n d i n g u n d e r the multiple s c h e d u l e o f food p r e s e n t a t i o n .
70
M c M I L L A N ET AL.
FIG. 7. Brain stem of pigeon No. 3 about four months after treatment with 1.75 mg/kg TMT. Chromatolytic and vacuolar changes were observed in many large brain stem neurons. Behavioral data for this bird are shown in Fig. 3. FIG. 8. Striatum griseum centrale (SGC) of pigeon No. 87 about three months after 1.75 mg/kg TMT. Pyknotic changes in some of the large neurons (arrows) in SGC were observed. Behavioral data for this bird are shown in Fig. 3.
FIG. 4. Hippocampus, pigeon, control. The hippocampus of a normal pigeon has a striatum consisting of medium size neurons (HC). FIG. 5. Hippocampus of pigeon No. 3 about four months after treatment with 1,75 mg/kg TMT. Total loss of hippocampal neurons in the hippocampus (HC) was evident. Behavioral data for this bird are shown in Fig. 3. FIG. 6. Brain stem, pigeon, control. The brain stem contains some large neurons (arrows) with prominent nuclei and Nissl substance.
R a t e s o f r e s p o n d i n g were greatly d e c r e a s e d in all 4 b i r d s d u r i n g the first few days a f t e r drug a d m i n i s t r a t i o n . S o m e r e c o v e r y o c c u r r e d in t h r e e o f the f o u r birds within a w e e k ; h o w e v e r , for bird 87 t h e r e was a s e c o n d d e c r e a s e in rate that became clear about 4 weeks after TMT administration. W i t h i n 6 w e e k s a f t e r the a d m i n i s t r a t i o n o f 1.75 mg/kg T M T , t w o birds h a d largely r e c o v e r e d from the effects of T M T , while two o t h e r s c o n t i n u e d to s h o w m a r k e d l y d e c r e a s e d r a t e s o f r e s p o n d i n g at the e n d o f t h e 8-week o b s e r v a t i o n period. Bird 53 was sacrificed for n e u r o p a t h o l o g i c a l o b s e r v a t i o n at the e n d o f the 7th w e e k . T h e quarter-life values for FI r e s p o n d i n g w e r e d e c r e a s e d for birds 52 a n d 87 for 3 d a y s a f t e r 1.75 mg/kg T M T , but few effects were o b s e r v e d t h e r e after. Bird 3 r e p r e s e n t s a particularly i n t e r e s t i n g case. This bird s t o p p e d r e s p o n d i n g t w o d a y s after T M T a d m i n i s t r a t i o n a n d m a d e n o f u r t h e r r e s p o n s e s d u r i n g the 8-week period. At this time the bird again w a s e x p o s e d to t h e t r a i n i n g p r o c e d u r e . Initially, t h e f e e d e r w a s a c t i v a t e d a u t o m a t i c a l l y several
TRIALKYLTINS AND PIGEON BEHAVIOR 1.0 MG/KG TRIETHYLTIN FIXED RATIO
175"
71
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175' 150'
5.0 MG/KG TRIETHYLTIN FIXED RATIO
75-
I00" 75' ~
50
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25'
50-
25 ~ ~)
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I
2
o
5 FIXED
O0
4
5
6
INTERVAL
175'
I
2
2501
3
4
~
5
6
FIXED INTERVAL\
§ 1 2°°1 e, '751
/\
\ ',,,
150' t~ o_
125
o_ 125 IO0
I00 "?5 50 25 O
I
2
5
4
5
6
FIG. 9. Effects of 1.0 mg/kg TET on responding under the mult schedule. Each point represents the rate for a bird during a single test session, beginning 3 hr after TET administration on day 1. Filled points fall more than two standard deviations from the mean. Bird 149 (O): Mean FR rate= 1.80 responses/sec. Mean FI rate=0.45 responses/sec. Bird 152 (~C): Mean FR rate= 1.40 responses/sec. Mean F1 rate=0.50 responses/sec. Bird 151 ([]): Mean FR rate=0.78 responses/sec. Mean FI rate=0.23 responses/sec.
175150-
5.6 MG/KG TRIETHYLTIN FIXED RATIO
I00" 75" l 50"
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25-
r~-
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r . . . . . . . . . . . . . . .
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5
4
r , , , , ....
5
6
FIXED INTERVAL
o,. 125I0075" 50" 25" I
2
3
4
I
2
3
4
5
6
WEEKS
WEEKS
5
6
WEEKS
FIG. 11. Effects of 5.6 mg/kg TET on responding under the mult schedule. Each point represents the rate for a bird during a single test session, beginning 3 hr after TET administration on day 1. Filled points fall more than two standard deviations from the mean. Bird 135 (O): Mean FR rate= 1.73 responses/sec. Mean FI rate=0.38 responses/sec. Bird 137 (A): Mean FR rate= 1.55 responses/sec. Mean FI rate=0.57 responses/sec. Bird 150 (D): Mean FR rate= 1.85 responses/sec. Mean FI rate=0.34 responses/sec.
FIG. 10. Effects of 3.0 mg/kg TET on responding under the mult schedule. Each point represents the rate for a bird during a single test session, beginning 3 hr after TET administration on day 1. Filled points fall more than two standard deviations from the mean. Bird 134 (O): Mean FR rate=l.43 responses/sec. Mean FR rate=0.69 responses/sec. Bird 136 (A): Mean FR rate=2.53 responses/sec. Mean FI rate=0.40 responses/sec. Bird 138 (IS]): Mean FR rate= 1.00 responses/sec. Mean FI rate=0.61 responses/sec.
times during the session and the bird was given the opportunity to eat. A presentation of food following a single response that o c c u r e d shortly thereafter, reinitiated key pecking and the bird was tested under a F R 20, FI 1-min schedule for the remainder of the session. On the next day the schedule was changed to F R 30 FI 5 min and the bird obtained food under the FI c o m p o n e n t , but rarely under the FR c o m p o n e n t . After a single session at a F R 30 FI 3 min schedule, the bird was tested under the F R 30 FI 5 min schedule and responding returned to near normal levels during the next week and was maintained for about a month prior to sacrifice o f the bird. Histopathological examination of animals treated with T M T revealed various loci of neuronal damage including the hippocampus (Figs. 4, 5), brain stem (Figs. 6, 7) and striatum griseum centrale (Fig. 8). T h e s e lesions were particularly severe in bird 3 and bird 87 and were only mildly involved in birds 52 and 53. The precise reason for such differences in brain neuropathology is unclear, but the overall observation that the degree of neuropathology was positively correlated with the magnitude of the behavioral deficit was consistent. Figure 9 shows the effects of 1.0 mg/kg T E T on rates of responding under the multiple schedule. All birds showed rate d e c r e a s e s under both schedule c o m p o n e n t s three hours after administration, but only bird 151 was still showing a d e c r e a s e d rate (FI c o m p o n e n t ) on the next day. This bird also s h o w e d s o m e rate-increasing effects, especially under the FI c o m p o n e n t during the third and fourth w e e k s after administration o f 1.0 mg/kg T E T . N o consistent effects were o b s e r v e d on the quarter-life values for FI responding. Figure 10 shows the effects of 3.0 mg/kg T E T on rates of responding. This dose nearly eliminated responding three hours after administration in all birds, and r e c o v e r y required
M c M I L L A N ET AL.
72
FIXED RATIO FIXED d-AMPHETAMINE
INTERVAL
3.0-
1.0
20"'::';::::::::~:~
06
0.4
1.0-
0.2 0o z 0
B A A'0.3
IO
3.0
B A A'0.3
3.0-
1.2-
2.0"
0.8-
w
w
FIG. 12. Brain stem of pigeon No. 151 about 4 months after treatment with 1.0 mg/kg TET. Aside from some remnants of myelin swelling in the white matter, no remarkable pathology was observed. Behavioral data for this bird are shown in Fig. 9.
z 0 o_
0.6l.O-
Drug Interactions Because the 1.0 mg/kg dose of TMT appeared to be close to a threshold dose for affecting responding under the mult FR 30 F1 5 schedule, this dose was given to a group of 4 pigeons that had previously been used to determine doseeffect curves for chlorpromazine, d-amphetamine and morphine. One month after administration of TMT, the doseeffect curves for these drugs were redetermined. After completion of the dose-effect curves, a second dose of TMT (1.5 mg/kg) was given and the dose-effect curves were redetermined, again beginning one month later. The effects of d-amphetamine, chlorpromazine and morphine before and after !.0 and 1.5 mg/kg T M T on responding under the multiple schedule are shown in Fig. 13. d-Amphetamine produced only rate-decreasing effects under both schedule components. After administration o f 1.0 mg/kg TMT, the d-amphetamine dose-effect curves were shifted to the right for both schedule components. No further changes were ob-
3.0
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MORPHINE 1.2-
3.0"
1 to 3 days depending on the bird. As with the lower dose, one bird (No. 136) showed some significant rate increases some weeks after 3.0 mg/kg TET, but for other birds only occasionally did points fall more than two standard deviations from the control mean. No consistent effects were observed on the quarter-life values for FI responding (Table 1). Figure 11 shows the effects of 5.6 mg/kg T E T on rates of responding. Responding was eliminated in all birds following this dose of TET. Rates of responding returned toward control levels more quickly under the FI component than under the FR component. From 2-5 days after 5.6 mg/kg TET, rates of responding under the FI component were well above control levels. During subsequent weeks only occasional points fell more than 2 standard deviations from the control mean. The quarter-life values for FI responding were decreased only for bird 150 during the first 3 days after 5.6 mg/kg T E T (Table 1). Few effects on quarter life were observed thereafter. Histopathological survey of brains from animals poisoned with TET revealed some edematous vacuolations of the white matter (myelinated fibers). Such an edematous condition was only minor and appeared to be reversible with time (Fig. 12).
1.0
CHLORPROMAZINE
I,O"
0.80.6-
Ii !~"
0.4-
I.O"
0.2" 0"
TTT
i
i
l
B A A' 0.5 1.0 3.0
i
O
I0
B A A ' O . 3 1.0 3.0 I0
MG/KG DOSE, LOG SCALE
FIG. 13. Effects of drugs in pigeons treated with TMT. Brackets show_+2 standard deviations around the mean before (B) TMT administration after (A) 1.0 mg/kg TMT and after (A') 1.5 mg/kg TMT. The shaded area shows the range of values during two determinations of the dose-effect curve before TMT exposure. Filled circles show dose-effect curves after 1.0 mg/kg TMT and open circles show curves after 1.5 mg/kg TMT. All data are based on means from 3 pigeons. served in these dose-effect curves after the 1.5 mg/kg dose of TMT. Low doses of chlorpromazine produced increases in rates of responding under the FI component of the schedule, while high doses decreased rates under both schedule components. The effects of chlorpromazine were not consistently affected by TMT administration. There was a statistically significant shift of the chlorpromazine dose-effect curve to the right (p<0.05) under the FR component after the 1.0 mg/kg dose of TMT, but this effect was not observed after the 1.5 mg/kg dose or under the FI component after either dose of TMT. Morphine produced rate-decreasing effects under both schedule components. TMT, at doses of 1.0 and 1.5 mg/kg, did not affect the morphine dose-effect curves. DISCUSSION
Initially, both alkyltin compounds produced decreases in rates of responding under the multiple schedule of food presentation. High doses of both alkyltin compounds affected quarter-life in some birds, but these effects only lasted a few days. The dose-effect curve for the rate-decreasing effects of TMT appeared to be steeper than that for TET. For example, 1.0 mg/kg TMT produced a threshold effect, while 1.75
T R I A L K Y L T I N S AND PIGEON BEHAVIOR
73
mg/kg produced effects that lasted for months in some birds. In contrast, the minimum effective dose of TET is about 1.0 mg/kg and birds recover from 5.6 mg/kg within a few days. TMT and TET also appear to differ in the duration of their effects on schedule-controlled behavior. For example, a dose of 1.75 mg/kg TMT and 3.0 to 5.6 mg/kg doses of TET produced similar initial effects on behavior, yet the birds given TET showed a recovery from this depression within a few days, while some birds given TMT continued to show severe effects for months. These behavioral data are consistent with the lack of neuronal lesions produced by the lower doses of TET in the pigeons as opposed to the more lasting brain damage produced by TMT. The potency of TMT in decreasing responding under the multiple FR FI schedule in pigeons is of approximately the same order of magnitude as that seen in mice. Wenger et al. [21] also found a 1.0 mg/kg dose of TMT to be close to a threshold dose in C57BL/6N mice, while 3.0 mg/kg markedly decreased response rates under both schedule components. BALB/c mice appeared to be slightly less sensitive to the effects of TMT. It appears that pigeons and mice are more sensitive to TMT than rats who generally require doses of 5-7 mg/kg to produce effects on schedule-controlled responding [13, 17, 19]. The neuropathology of TMT and TET in pigeons is consistent with that found in mammalian brains, TMT being neurotoxic and TET myelinotoxic. When the neuropathology of TMT is compared with its behavioral effects, some correlations begin to occur. For example, the 0.3 mg/kg dose of TMT did not produce effects on schedule-controlled behavior, nor did this dose produce effects detectable by light microscopy. The 1.0 mg/kg TMT dose, which was a threshold dose for producing effects on schedule-controlled responding, produced neuronal necrosis in the striatum griseum centrale and neuronal loss in the hippocampus and brain stem. Higher doses of TMT produced progressively more extensive lesions and more marked behavioral effects. Although it is not yet possible to correlate any specific behavioral change with any specific lesion site, it does appear that the morphological and behavioral changes in the pigeon began to occur at about the same time and increase in severity with increasing doses. Figure 3 showed that birds subjected to a 1.75 mg/kg dose
of TMT showed large decreases in rates of responding 3 hr after TMT administration. After a few days, some birds began to recover, while other did not. Since all birds given 1.75 mg/kg TMT showed neuronal loss in the hippocampus and striatum griseum centrale, it may be that the acute (beginning 3 hr after administration) effects of TMT are not related to the long-term neuronal loss produced by TMT, but rather are a function of other acute toxic effects of TMT. In this respect, pigeons appear to differ from mice in that the maximum effect of TMT in mice does not become apparent until 2 or 3 days after TMT administration [21]. It is particularly tempting to speculate about why one bird, whose responding had been totally eliminated by 1.75 mg/kg TMT for 8 weeks, could easily be retrained to his control performance in a few days by presenting him with free food to reinitiate key pecking. It seems unlikely that the drug interferred with long-term memory processes since the retraining was accomplished so rapidly. It is also of interest to compare the difference in extent of pathological involvement in birds 3 and 87 versus birds 52 and 53 which were treated with the same dose of TMT. The birds with the more extensive neuropathological involvement showed the more severe and prolonged behavioral deficits. Thus, the result of the morphological and behavioral studies was consistent. Treatment of pigeons with TMT inconsistently modified the effects of chlorpromazine on schedule-controlled behavior and did not modify the effect of morphine on schedulecontrolled behavior; however, the dose-response curve for d-amphetamine was shifted to the right in birds treated with TMT. It seems unlikely that this shift to the right in the dose-effect curve represents a cross-tolerance from TMT to d-amphetamine. It is more likely that the lesions produced by TMT in pigeons somehow interfere with the expression of the drug effect, although in what way is not clear.
ACKNOWLEDGEMENTS Although the research described in this article was funded in part by the United States EPA under assistance agreement number R809452 to the University of Arkansas for Medical Sciences, it has not been subjected to the Agency's peer and administrative review and, therefore, does not reflect the view of the Agency and no official endorsement should be inferred.
REFERENCES 1. Brown, A. W., W. N. Aldridge, B. W. Street and R. D. Verschoyle. The behavioral and neuropathologic sequelae of intoxication by trimethyltin compounds in the rat. Am J Pathol 97: 5%81, 1979. 2. Chang, L. W., T. M. Tiemeyer, G. R. Wenger and D. E. McMillan. Neuropathology of mouse hippocampus in acute trimethyltin intoxication. Neurobehav Toxicol Teratol 4: 14%256, 1982. 3. DeHaven, D. L., M. J. Wayner and F. C. Barone. Effects of triethyltin on ingestive behavior at ad lib, reduced, and recovered body weight. Neurobehav Toxicol Teratol 4: 217-223, 1982. 4. Dietz, D. D., D. E. McMillan, L. D. Grant and C. A. Kimmel. Effects of lead on temporally spaced responding in rats. Drug Chem Toxicol 1: 401-419, 1978. 5. Ferster, C. B. and B. F. Skinner. Schedules o f Reinfi~rcement. New York: Appleton-Century-Crofts, 1957. 6. Gage, M. I., R. Rhoderick, R. J. Pettinelli, J. D. Edwards, E. T. Puckett and L. W. Reiter. Operant behavior of rats postnatally exposed to triethyltin (TET). Toxicologist 1: 45, 1981.
7. Goldstein, A. Biostatistics: An Introductory Text. New York: MacMillan Co., 1964. 8. Gollub, L. R. The relations among measures of performance of fixed-interval schedules. J Exp Anal Behav 7: 337-343, 1964. 9. Hughes, J. A. and S. B. Sparber. d-Amphetamine unmasks postnatal consequences of exposure to methylmercury in utero: Methods for studying behavioral teratogenesis. Pharmacol Biochem Behav 9: 365-375, 1978. 10. Leander, J. D. and B. A. Gau. Flavor aversions rapidly produced by inorganic lead and triethyl tin. Neurotoxicology 1: 635-642, 1980. 11. McMillan, D. E. and J. D. Leander. Modification of baseline operant behavior by drugs. In: Behavioral Pharmacology, edited by S. D. Glick and J. Goldfarb. St Louis: Mosby Co., 1976, pp. 85-139. 12. McMillan, D. E. and A. T Miller. Interactions between carbon monoxide and d-amphetamine, or pentobarbital on schedulecontrolled behavior. Environ Res 8: 53-63, 1974. 13. McMillan, D. E. and G. R. Wenger. Neurobehavioral toxicology of trialkyltins. Pharrnacol Rev 37: 365-379, 1985.
74
14. Organization for Economic Co-Operation and Development. Guidelines for the Testing of Chemicals. Paris, 1981. 15. Reiter, L. W., K. Kidd, G. Heavener and P. Rupert. Behavioral toxicity of acute and subacute exposure to triethyltin in the rat. Neurotoxicology 2: 97-112, 1980. 16. Squibb, R. E., N. G. Carmichael and H. A. Tilson. Behavioral and neuromorphological effects of triethyl tin bromide in adult rats. Toxicol Appl Pharmacol 55: 188-197, 1980. 17. Swartzwelder, H. S., R. S. Dyer, W. Holahan and R. D. Myers. Activity changes in rats following acute trimethyltin exposure. Neurotoxicology 2: 58%593, 1981.
McMILLAN ET AL. 18. U.S. Environmental Protection Agency. Proposed guidelines for registering pesticides in the United States: Hazard evaluation: Humans and domestic animals. Fed Reg 43: 37336-37403, 1978. 19. Walsh, T. J., J. M. Gallagher, E. Bostock and R. S. Dyer. Trimethyltin impairs retention of a passive avoidance task. Neurobehav Toxicol 4: 163-168, 1982. 20. Wenger, G. R., D. E. McMillan and L. W. Chang. Behavioral toxicology of acute trimethyltin exposure in the mouse. Neurobehav Toxicol Teratol 4: 157-161, 1982. 21. Wenger, G. R., D. E. McMillan and L. W. Chang. Behavioral effects of trimethyltin in two strains of mice, I1. Multiple fixed ratio, fixed interval. Toxicol Appl Pharmacol 73: 89-96, 1984.
0892-0362/87$3.00 + .00
Effects of Trialkyltins on the Schedule-Controlled Behavior of the Pigeon D. E. M c M I L L A N , * G. R. W E N G E R , * M. J. B R O C C O , .1 S. O. I D E M U D I A * A N D L. W. C H A N G * t
*Department of Pharmacology and Interdisciplinary Toxicology and tDepartment of Pathology University of Arkansas for Medical Sciences, 4301 W. Markham Street, Little Rock, AR 72205 R e c e i v e d 11 F e b r u a r y 1985 McMILLAN, D. E., G. R. WENGER, M. J. BROCCO, S. O. 1DEMUDIA AND L. W. CHANG. Effects oftrialkyltins on the schedule-controlled behavior of the pigeon. NEUROTOXICOL TERATOL 9(1) 67-74, 1987.--Male White Carneaux pigeons trained to respond for food under a multiple fixed-ratio fixed-interval schedule of reinforcement were given single injections of trimethyltin (TMT), or triethyltin (TET). A dose of 0.3 mg/kg TMT produced no effect on behavior, while a 1.0 mg/kg dose was a threshold dose and 1.75 mg/kg produced behavioral changes that persisted for months in some birds. TMT produced effects on responding under the multiple schedule at approximately the same doses that produce neuronal damage in the hippocampus and the brain stem of the pigeon. Higher doses given to untrained birds produced signs of extensive neurological damage. A dose of 1.0 mg/kg of TET decreased rates of responding under both schedule components three hours after administration, but behavior usually had recovered by the next day. Doses of 3.0 and 5.6 mg/kg had similar effects, but responding did not recover for several days. Some birds showed significant rate increases, especially under the fixed-interval component several days to several weeks after TET administration. Doses greater than 10 mg/kg TET were lethal. Dose-effect curves for the effects of d-amphetamine, chlorpromazine and morphine on responding under the multiple schedule were determined for some birds before and one month after 1.0 and 1.5 mg/kg of TMT. TMT shifted the dose-effect curve for d-amphetamine to the right, but it did not produce systematic changes in the dose-effect curves for morphine and chlorpromazine. Trialkyltins
Pigeons
Schedule-controlled behavior
METHOD
BECAUSE organic tin compounds have been used widely in industry and because they produce specific localized lesions in the brain [1, 2, 12], there has been considerable interest in the behavioral toxicology of these compounds [1, 6, 10, 13, 16, 20, 21]. Although operant behavior has been used to describe the effects of many chemicals on behavior [1 I], only a few studies have reported on the effects of alkyltins on schedule-controlled behavior [17,21]. The present study provides a comparison of the effects of trimethyltin (TMT) and triethyltin (TET) on responding maintained by a multiple schedule of food presentation in the pigeon. The pigeon was chosen because there is a large literature on the effects of drugs and toxic chemicals on schedule-controlled behavior in pigeons and because birds have been suggested as subjects in several guidelines for neurotoxicity testing [14,18]. Pharmacological challenge has been shown to be useful for evaluating exposure to many types of toxicants [3, 4, 12] and in some instances has proven exceedingly sensitive in detecting behavioral effects [9]. Therefore, the effects of d-amphetamine, chlorpromazine and morphine were determined before and after TMT administration.
Animals A total of 21 adult male White Carneaux pigeons (Palmetto Pigeon Plant, Sumter, SC), used to measure the effects of alkyltins on schedule-controlled behavior, were maintained at 80% of their free-feeding weights throughout all experiments. Their 80% body weights ranged from 410 to 518 g. Birds were fed mixed grain (Ralston-Purina pigeon diet). Crushed oyster shell was freely available in home cages.
Materials and Dosing TMT hydrochloride solutions were prepared for administration as 0.3, 1.0 and 1.75 mg/ml and TET sulfate solutions as 1.0, 3.0 and 5.6 mg/ml. Doses were calculated as the salts. Distilled water was given as a control injection and distilled water served as the solvent for both alkyltin salts. Injection volumes were 0.1 ml/100 g of body weight. All injections were given 3 hours before behavioral testing by the intramuscular route.
1Present address: Institut de Recherche Servier, 14 Due du Val d'Or, Suresnes, 92150, France.
67
M c M I L L A N ET AL.
68 03
t.O MG/KG T R I M E T H Y L T I N
MG/KG TRIMETHYLTIN
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I001
5O 0
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2 3 FIXED INTERVAL
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2003 175"
F-
150"
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125 l
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4
150"
75'
0
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RATIO e------=
25]
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FIXED RATIO
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FIG. 1. Effects of 0.3 mg/kg TMT on responding under the mult schedule. Each point represents the rate for a bird during a single test session, beginning 3 hr after TMT administration on day 1. Filled points fall more than two standard deviations from the mean. Bird 126 (©): Mean FR rate=2.30 responses/sec. Mean FI rate=0.33 responses/sec. Bird 127 (A): Mean FR rate=2.05 responses/sec. Mean FI rate=0.68 responses/sec. Bird 128 (El): Mean FR rate=2.53 responses/sec. Mean FI rate=0.19 responses/sec.
In drug interaction experiments, d-amphetamine sulfate, chlorpromazine hydrochloride and morphine sulfate were given intramuscularly 10 min before the session. Two birds received an ascending dosage series and one a descending dosage series. After determination of the dose-effect curves with two observations at each dose level in each of 3 birds, 1.0 mg/kg TMT was given and behavioral testing was continued for one month, after which the dose-effect curve was redetermined for each of the 3 drugs. After redetermination of the dose-effect curves, a. 1.5 mg/kg dose of TMT was given and after an additional month of behavioral testing the dose-effect curves for the 3 drugs were redetermined again.
Apparatus for Behavioral Testing Behavioral testing was conducted in two sound- and light-attenuating chambers similar to those described by Ferster and Skinner [5]. The chamber was illuminated by a 28 V DC light and contained a translucent plastic response key (2.0 cm in diameter) which was centrally mounted in the chamber wall 22 cm above a wire-mesh floor. The response key could be transilluminated by red or blue lights. The minimum force required to operate the key was about 0. I N. Centered below the key and 6 cm above the floor was a rectangular opening through which the pigeon could be given access to mixed grain. During access to grain all lights except those illuminating the feeder were off. Relay programming and recording apparatus housed in the adjacent room controlled the delivery of grain and recorded the pattern of key pecking. A masking noise was supplied by a speaker mounted inside the chamber.
Procedure Pigeons performed Monday through Friday under a multiple fixed-ratio, fixed-interval schedule (mult FR FI) of food
5 ....
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WEEKS
WEEKS
FIG. 2. Effects of 1.0 mg/kg TMT on responding under the mult schedule. Each point represents the rate for a bird during a single test session, beginning 3 hr after TMT administration on day 1. Filled points fall more than two standard deviations from the mean. Bird 1 (©): Mean FR rate= 1.56 responses/sec. Mean FI rate=0.43 responses/sec. Bird 4 (A): Mean FR rate=5.04 response/sec. Mean FI rate=l.28 responses/sec. Bird 101 ([]): Mean FR rate=2.65 responses/sec. Mean F1 rate=0.95 responses/sec. Bird 102 (~): Mean FR rate= 1.68 response/sec. Mean F1 rate=0.58 response/sec. Bird 106 (V): Mean FR rate=2.20 responses/sec. Mean FI rate=0.46 responses/sec. Birds 1 and 4 received additional injections of TMT beginning in the third week so their data are shown for only the first two weeks after TMT administration.
presentation. In the presence of a blue light, the 30th key peck (FR 30) produced 4-sec access to grain. In the presence of a red light, the first response after 5 min (F! 5) produced 4-sec access to grain. The FR and FI components alternated after each food presentation. If a bird failed to respond within 30 sec after 5 min had elapsed during the FI component, the schedule changed to the FR component. If the bird failed to make 30 responses within 40 sec during the FR component, the schedule changed to the F1 component. Sessions were started in the FR component I0 rain after the pigeon was placed in the chamber and were terminated after 6 presentations of each schedule component. Upon termination of the behavioral experiments the pigeons were sacrificed for examination by light microscopy. At sacrifice, pigeons were anesthetized with pentobarbital and were perfused intracardially with saline solution followed with 2.5% buffered glutaraldehyde. Animals were then decapitated and the brains removed carefully, further fixed in Bovins-formalin fixative, and prepared for histopathological examination.
Data Analysis For pigeons responding under the multiple schedule, the rates of FR and FI responding were calculated daily. The quarter life (a measure of the pattern of FI responding) was determined according to the method of Gollub [8]. Data from at least 8 control sessions, before some of which saline injections were given, were used to determine control means and standard deviations. Points falling more than two standard deviations from the control mean after TMT or TET
TRIALKYLTINS
AND PIGEON BEHAVIOR
69
TABLE 1
1.75 MG/KG TRIMETHYLTIN FIXED RATIO
EFFECTS OF TMT AND T E T ON Q U A R T E R L I F E (QL EXPRESSED AS PERCENT)
,,ours Af, er Alkyltin Drug
Dose (mg/kg)
TMT
1.0
TMT
TET
TET
1.75
3.0
5.6
Bird
Control QL (mean _+ 2 SDs)
1 4 101 102 106
41 41 36 31 37
_+ 73 _+ 69 _+ 66 _+ 59 _+ 73
3 53 52 87
40 40 49 54
+ 72 _+ 67 _+ 75 _+ 70
3
27
51
74
45 41
43 53
55 49
43 47
44
49
47
37
X
X
x4,
49
46
40
56
X
X
44
x x
29 36
485 51+
0205 .
134 136 138
27 + 57 36 + 67 32 _+ 50
x
48
48
50
x
29~
37
47
x
33
51 i'
51'~
135 137 150
38 _+ 68 28 _+ 58 42 _+ 52
x x x
39 39 185
42 33 395
42 33 35,~
x: Indicates too few responses occurred to determine a reliable quarter life. J,=Decreased quarter life (more than 2 SDs below the control mean). 1'=Increased quarter life (more than 2 SDs above the control mean).
a d m i n i s t r a t i o n for individual birds were c o n s i d e r e d to be significant. In the drug i n t e r a c t i o n s t u d i e s , g r o u p m e a n s a n d s t a n d a r d d e v i a t i o n s w e r e c a l c u l a t e d for c o n t r o l sessions. T h e effects o f d - a m p h e t a m i n e , c h l o r p r o m a z i n e a n d m o r p h i n e w e r e considered to be significantly different f r o m the c o n t r o l rate w h e n t h e m e a n rate o f r e s p o n d i n g a f t e r a dose was m o r e t h a n 2 s t a n d a r d d e v i a t i o n s f r o m the c o n t r o l m e a n . W h e n d o s e - e f f e c t c u r v e s w e r e c o m p a r e d for d r u g s g i v e n before a n d a f t e r T M T a d m i n i s t r a t i o n , t h e d o s e - e f f e c t c u r v e for e a c h drug was d e t e r m i n e d t w i c e a n d the range was plotted to s h o w t h a t the d o s e - e f f e c t c u r v e was n o t variable. T h e d o s e - e f f e c t c u r v e s for e a c h d r u g a f t e r T M T w e r e c o m p a r e d w i t h the m e a n d o s e - e f f e c t c u r v e b e f o r e T M T with the o b s e r v a t i o n s at e a c h d o s e paired (before a n d after) for a n a l y s i s o f t h e d o s e - e f f e c t c u r v e d i f f e r e n c e s b y the signed r a n k s test [7]. RESULTS
Visual Observation o f Behavioral Effects A t a d o s e of 1.0 mg/kg T M T , no g r o s s b e h a v i o r a l effects w e r e o b s e r v e d . A d o s e o f 1.75 m g / k g p r o d u c e d d e c r e a s e d r e a c t i o n to h a n d l i n g a n d ataxia in 3 o f 4 b i r d s w i t h r e c o v e r y after about 3 days; however, one of these birds showed a d e c r e a s e d food i n t a k e a n d p o s t u r a l c h a n g e s w h i c h lasted for several m o n t h s . A single 3 mg/kg d o s e o f T M T p r o d u c e d t r e m o r s , a t a x i a a n d d e c r e a s e d r e a c t i o n to h a n d l i n g w i t h i n a few h o u r s time. T h e s e birds w e r e sacrificed 24 h o u r s a f t e r T M T a d m i n i s t r a t i o n . Birds g i v e n d o s e s a b o v e 3.0 mg/kg T M T died w i t h i n 24 hr. A t a 1.0 mg/kg d o s e o f T E T , no g r o s s b e h a v i o r a l effects
.
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.
5 FIXED
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~INTERVAL
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WEEKS
FIG. 3. Effects of 1.75 mg/kg TMT on responding under the mult schedule. Each point represents the rate for a bird during a single test session, beginning 3 hr after TMT administration on day 1. Filled points fall more than two standard deviations from the mean. Bird 3 ((3): Mean FR rate=0.65 responses/sec. Mean F1 rate=0.33 responses/sec. Bird 52 (A): Mean FR rate=l.26 responses/sec. Mean FI rate=0.23 responses/sec. Bird 53 (IS]): Mean FR rate= 1.38 responses/sec. Mean F1 rate=0.14 responses/sec. Bird 87 (O): Mean FR rate=l.95 responses/sec. Mean FI rate=0.34 responses/sec. Bird 53 was sacrificed for histological examination at the end of the 7th week after TMT administration.
w e r e o b s e r v e d . T h e 3.0 mg/kg d o s e p r o d u c e d t r e m o r s , a t a x i a a n d m u s c l e rigidity, but by 24 h o u r s a f t e r the injection, t h e birds no l o n g e r s h o w e d a n y g r o s s s y m p t o m s . T h e 5.6 mg/kg d o s e c a u s e d the birds to collapse w i t h a high d e g r e e o f muscle rigidity. T h e r e w a s a rapid o n s e t ( a b o u t l0 min) o f t r e m ors a n d the birds w e r e u n a b l e to walk or fly. T h e b i r d s app e a r e d to b e n o r m a l at 24 h o u r s , e x c e p t t h a t o n e bird s h o w e d a r e d u c e d food intake at this time.
Effects on Mult FI FR Performance T h e effects of 0.3 mg/kg T M T o n r e s p o n d i n g u n d e r the mult F R FI s c h e d u l e are s h o w n in Fig. 1 for 3 individual birds. A l t h o u g h r a t e s o f r e s p o n d i n g o c c a s i o n a l l y fell m o r e t h a n 2 s t a n d a r d d e v i a t i o n s f r o m the m e a n c o n t r o l , this occ u r r e d only 7 t i m e s in 120 o b s e r v a t i o n s d u r i n g a 4 - w e e k period, indicating t h a t 0.3 mg/kg T M T p r o d u c e d n o signific a n t effect o n r a t e s o f r e s p o n d i n g u n d e r the multiple s c h e d u l e . T h e 0.3 mg/kg d o s e o f T M T did n o t affect the quarter-life values for FI r e s p o n d i n g . Figure 2 s h o w s t h e effects o f 1.0 mg/kg T M T o n rates of r e s p o n d i n g u n d e r the multiple s c h e d u l e o f food p r e s e n t a t i o n . B o t h i n c r e a s e s a n d d e c r e a s e s in rate o f r e s p o n d i n g were obs e r v e d a l t h o u g h the effects were small a n d were n o t obs e r v e d e v e r y day. T h e s e small effects o n r a t e s o f r e s p o n d i n g were o b s e r v e d d u r i n g s o m e s e s s i o n s for t h e e n t i r e 8-week period d u r i n g w h i c h o b s e r v a t i o n s were m a d e . T h e 1.0 mg/kg d o s e of T M T did not affect the quarter-life values for FI r e s p o n d i n g (Table 1). Figure 3 s h o w s t h e effects o f 1.75 mg/kg T M T o n r a t e s of r e s p o n d i n g u n d e r the multiple s c h e d u l e o f food p r e s e n t a t i o n .
70
M c M I L L A N ET AL.
FIG. 7. Brain stem of pigeon No. 3 about four months after treatment with 1.75 mg/kg TMT. Chromatolytic and vacuolar changes were observed in many large brain stem neurons. Behavioral data for this bird are shown in Fig. 3. FIG. 8. Striatum griseum centrale (SGC) of pigeon No. 87 about three months after 1.75 mg/kg TMT. Pyknotic changes in some of the large neurons (arrows) in SGC were observed. Behavioral data for this bird are shown in Fig. 3.
FIG. 4. Hippocampus, pigeon, control. The hippocampus of a normal pigeon has a striatum consisting of medium size neurons (HC). FIG. 5. Hippocampus of pigeon No. 3 about four months after treatment with 1,75 mg/kg TMT. Total loss of hippocampal neurons in the hippocampus (HC) was evident. Behavioral data for this bird are shown in Fig. 3. FIG. 6. Brain stem, pigeon, control. The brain stem contains some large neurons (arrows) with prominent nuclei and Nissl substance.
R a t e s o f r e s p o n d i n g were greatly d e c r e a s e d in all 4 b i r d s d u r i n g the first few days a f t e r drug a d m i n i s t r a t i o n . S o m e r e c o v e r y o c c u r r e d in t h r e e o f the f o u r birds within a w e e k ; h o w e v e r , for bird 87 t h e r e was a s e c o n d d e c r e a s e in rate that became clear about 4 weeks after TMT administration. W i t h i n 6 w e e k s a f t e r the a d m i n i s t r a t i o n o f 1.75 mg/kg T M T , t w o birds h a d largely r e c o v e r e d from the effects of T M T , while two o t h e r s c o n t i n u e d to s h o w m a r k e d l y d e c r e a s e d r a t e s o f r e s p o n d i n g at the e n d o f t h e 8-week o b s e r v a t i o n period. Bird 53 was sacrificed for n e u r o p a t h o l o g i c a l o b s e r v a t i o n at the e n d o f the 7th w e e k . T h e quarter-life values for FI r e s p o n d i n g w e r e d e c r e a s e d for birds 52 a n d 87 for 3 d a y s a f t e r 1.75 mg/kg T M T , but few effects were o b s e r v e d t h e r e after. Bird 3 r e p r e s e n t s a particularly i n t e r e s t i n g case. This bird s t o p p e d r e s p o n d i n g t w o d a y s after T M T a d m i n i s t r a t i o n a n d m a d e n o f u r t h e r r e s p o n s e s d u r i n g the 8-week period. At this time the bird again w a s e x p o s e d to t h e t r a i n i n g p r o c e d u r e . Initially, t h e f e e d e r w a s a c t i v a t e d a u t o m a t i c a l l y several
TRIALKYLTINS AND PIGEON BEHAVIOR 1.0 MG/KG TRIETHYLTIN FIXED RATIO
175"
71
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175' 150'
5.0 MG/KG TRIETHYLTIN FIXED RATIO
75-
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O0
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INTERVAL
175'
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FIG. 9. Effects of 1.0 mg/kg TET on responding under the mult schedule. Each point represents the rate for a bird during a single test session, beginning 3 hr after TET administration on day 1. Filled points fall more than two standard deviations from the mean. Bird 149 (O): Mean FR rate= 1.80 responses/sec. Mean FI rate=0.45 responses/sec. Bird 152 (~C): Mean FR rate= 1.40 responses/sec. Mean F1 rate=0.50 responses/sec. Bird 151 ([]): Mean FR rate=0.78 responses/sec. Mean FI rate=0.23 responses/sec.
175150-
5.6 MG/KG TRIETHYLTIN FIXED RATIO
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FIXED INTERVAL
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2
3
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3
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WEEKS
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5
6
WEEKS
FIG. 11. Effects of 5.6 mg/kg TET on responding under the mult schedule. Each point represents the rate for a bird during a single test session, beginning 3 hr after TET administration on day 1. Filled points fall more than two standard deviations from the mean. Bird 135 (O): Mean FR rate= 1.73 responses/sec. Mean FI rate=0.38 responses/sec. Bird 137 (A): Mean FR rate= 1.55 responses/sec. Mean FI rate=0.57 responses/sec. Bird 150 (D): Mean FR rate= 1.85 responses/sec. Mean FI rate=0.34 responses/sec.
FIG. 10. Effects of 3.0 mg/kg TET on responding under the mult schedule. Each point represents the rate for a bird during a single test session, beginning 3 hr after TET administration on day 1. Filled points fall more than two standard deviations from the mean. Bird 134 (O): Mean FR rate=l.43 responses/sec. Mean FR rate=0.69 responses/sec. Bird 136 (A): Mean FR rate=2.53 responses/sec. Mean FI rate=0.40 responses/sec. Bird 138 (IS]): Mean FR rate= 1.00 responses/sec. Mean FI rate=0.61 responses/sec.
times during the session and the bird was given the opportunity to eat. A presentation of food following a single response that o c c u r e d shortly thereafter, reinitiated key pecking and the bird was tested under a F R 20, FI 1-min schedule for the remainder of the session. On the next day the schedule was changed to F R 30 FI 5 min and the bird obtained food under the FI c o m p o n e n t , but rarely under the FR c o m p o n e n t . After a single session at a F R 30 FI 3 min schedule, the bird was tested under the F R 30 FI 5 min schedule and responding returned to near normal levels during the next week and was maintained for about a month prior to sacrifice o f the bird. Histopathological examination of animals treated with T M T revealed various loci of neuronal damage including the hippocampus (Figs. 4, 5), brain stem (Figs. 6, 7) and striatum griseum centrale (Fig. 8). T h e s e lesions were particularly severe in bird 3 and bird 87 and were only mildly involved in birds 52 and 53. The precise reason for such differences in brain neuropathology is unclear, but the overall observation that the degree of neuropathology was positively correlated with the magnitude of the behavioral deficit was consistent. Figure 9 shows the effects of 1.0 mg/kg T E T on rates of responding under the multiple schedule. All birds showed rate d e c r e a s e s under both schedule c o m p o n e n t s three hours after administration, but only bird 151 was still showing a d e c r e a s e d rate (FI c o m p o n e n t ) on the next day. This bird also s h o w e d s o m e rate-increasing effects, especially under the FI c o m p o n e n t during the third and fourth w e e k s after administration o f 1.0 mg/kg T E T . N o consistent effects were o b s e r v e d on the quarter-life values for FI responding. Figure 10 shows the effects of 3.0 mg/kg T E T on rates of responding. This dose nearly eliminated responding three hours after administration in all birds, and r e c o v e r y required
M c M I L L A N ET AL.
72
FIXED RATIO FIXED d-AMPHETAMINE
INTERVAL
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FIG. 12. Brain stem of pigeon No. 151 about 4 months after treatment with 1.0 mg/kg TET. Aside from some remnants of myelin swelling in the white matter, no remarkable pathology was observed. Behavioral data for this bird are shown in Fig. 9.
z 0 o_
0.6l.O-
Drug Interactions Because the 1.0 mg/kg dose of TMT appeared to be close to a threshold dose for affecting responding under the mult FR 30 F1 5 schedule, this dose was given to a group of 4 pigeons that had previously been used to determine doseeffect curves for chlorpromazine, d-amphetamine and morphine. One month after administration of TMT, the doseeffect curves for these drugs were redetermined. After completion of the dose-effect curves, a second dose of TMT (1.5 mg/kg) was given and the dose-effect curves were redetermined, again beginning one month later. The effects of d-amphetamine, chlorpromazine and morphine before and after !.0 and 1.5 mg/kg T M T on responding under the multiple schedule are shown in Fig. 13. d-Amphetamine produced only rate-decreasing effects under both schedule components. After administration o f 1.0 mg/kg TMT, the d-amphetamine dose-effect curves were shifted to the right for both schedule components. No further changes were ob-
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MORPHINE 1.2-
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1 to 3 days depending on the bird. As with the lower dose, one bird (No. 136) showed some significant rate increases some weeks after 3.0 mg/kg TET, but for other birds only occasionally did points fall more than two standard deviations from the control mean. No consistent effects were observed on the quarter-life values for FI responding (Table 1). Figure 11 shows the effects of 5.6 mg/kg T E T on rates of responding. Responding was eliminated in all birds following this dose of TET. Rates of responding returned toward control levels more quickly under the FI component than under the FR component. From 2-5 days after 5.6 mg/kg TET, rates of responding under the FI component were well above control levels. During subsequent weeks only occasional points fell more than 2 standard deviations from the control mean. The quarter-life values for FI responding were decreased only for bird 150 during the first 3 days after 5.6 mg/kg T E T (Table 1). Few effects on quarter life were observed thereafter. Histopathological survey of brains from animals poisoned with TET revealed some edematous vacuolations of the white matter (myelinated fibers). Such an edematous condition was only minor and appeared to be reversible with time (Fig. 12).
1.0
CHLORPROMAZINE
I,O"
0.80.6-
Ii !~"
0.4-
I.O"
0.2" 0"
TTT
i
i
l
B A A' 0.5 1.0 3.0
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MG/KG DOSE, LOG SCALE
FIG. 13. Effects of drugs in pigeons treated with TMT. Brackets show_+2 standard deviations around the mean before (B) TMT administration after (A) 1.0 mg/kg TMT and after (A') 1.5 mg/kg TMT. The shaded area shows the range of values during two determinations of the dose-effect curve before TMT exposure. Filled circles show dose-effect curves after 1.0 mg/kg TMT and open circles show curves after 1.5 mg/kg TMT. All data are based on means from 3 pigeons. served in these dose-effect curves after the 1.5 mg/kg dose of TMT. Low doses of chlorpromazine produced increases in rates of responding under the FI component of the schedule, while high doses decreased rates under both schedule components. The effects of chlorpromazine were not consistently affected by TMT administration. There was a statistically significant shift of the chlorpromazine dose-effect curve to the right (p<0.05) under the FR component after the 1.0 mg/kg dose of TMT, but this effect was not observed after the 1.5 mg/kg dose or under the FI component after either dose of TMT. Morphine produced rate-decreasing effects under both schedule components. TMT, at doses of 1.0 and 1.5 mg/kg, did not affect the morphine dose-effect curves. DISCUSSION
Initially, both alkyltin compounds produced decreases in rates of responding under the multiple schedule of food presentation. High doses of both alkyltin compounds affected quarter-life in some birds, but these effects only lasted a few days. The dose-effect curve for the rate-decreasing effects of TMT appeared to be steeper than that for TET. For example, 1.0 mg/kg TMT produced a threshold effect, while 1.75
T R I A L K Y L T I N S AND PIGEON BEHAVIOR
73
mg/kg produced effects that lasted for months in some birds. In contrast, the minimum effective dose of TET is about 1.0 mg/kg and birds recover from 5.6 mg/kg within a few days. TMT and TET also appear to differ in the duration of their effects on schedule-controlled behavior. For example, a dose of 1.75 mg/kg TMT and 3.0 to 5.6 mg/kg doses of TET produced similar initial effects on behavior, yet the birds given TET showed a recovery from this depression within a few days, while some birds given TMT continued to show severe effects for months. These behavioral data are consistent with the lack of neuronal lesions produced by the lower doses of TET in the pigeons as opposed to the more lasting brain damage produced by TMT. The potency of TMT in decreasing responding under the multiple FR FI schedule in pigeons is of approximately the same order of magnitude as that seen in mice. Wenger et al. [21] also found a 1.0 mg/kg dose of TMT to be close to a threshold dose in C57BL/6N mice, while 3.0 mg/kg markedly decreased response rates under both schedule components. BALB/c mice appeared to be slightly less sensitive to the effects of TMT. It appears that pigeons and mice are more sensitive to TMT than rats who generally require doses of 5-7 mg/kg to produce effects on schedule-controlled responding [13, 17, 19]. The neuropathology of TMT and TET in pigeons is consistent with that found in mammalian brains, TMT being neurotoxic and TET myelinotoxic. When the neuropathology of TMT is compared with its behavioral effects, some correlations begin to occur. For example, the 0.3 mg/kg dose of TMT did not produce effects on schedule-controlled behavior, nor did this dose produce effects detectable by light microscopy. The 1.0 mg/kg TMT dose, which was a threshold dose for producing effects on schedule-controlled responding, produced neuronal necrosis in the striatum griseum centrale and neuronal loss in the hippocampus and brain stem. Higher doses of TMT produced progressively more extensive lesions and more marked behavioral effects. Although it is not yet possible to correlate any specific behavioral change with any specific lesion site, it does appear that the morphological and behavioral changes in the pigeon began to occur at about the same time and increase in severity with increasing doses. Figure 3 showed that birds subjected to a 1.75 mg/kg dose
of TMT showed large decreases in rates of responding 3 hr after TMT administration. After a few days, some birds began to recover, while other did not. Since all birds given 1.75 mg/kg TMT showed neuronal loss in the hippocampus and striatum griseum centrale, it may be that the acute (beginning 3 hr after administration) effects of TMT are not related to the long-term neuronal loss produced by TMT, but rather are a function of other acute toxic effects of TMT. In this respect, pigeons appear to differ from mice in that the maximum effect of TMT in mice does not become apparent until 2 or 3 days after TMT administration [21]. It is particularly tempting to speculate about why one bird, whose responding had been totally eliminated by 1.75 mg/kg TMT for 8 weeks, could easily be retrained to his control performance in a few days by presenting him with free food to reinitiate key pecking. It seems unlikely that the drug interferred with long-term memory processes since the retraining was accomplished so rapidly. It is also of interest to compare the difference in extent of pathological involvement in birds 3 and 87 versus birds 52 and 53 which were treated with the same dose of TMT. The birds with the more extensive neuropathological involvement showed the more severe and prolonged behavioral deficits. Thus, the result of the morphological and behavioral studies was consistent. Treatment of pigeons with TMT inconsistently modified the effects of chlorpromazine on schedule-controlled behavior and did not modify the effect of morphine on schedulecontrolled behavior; however, the dose-response curve for d-amphetamine was shifted to the right in birds treated with TMT. It seems unlikely that this shift to the right in the dose-effect curve represents a cross-tolerance from TMT to d-amphetamine. It is more likely that the lesions produced by TMT in pigeons somehow interfere with the expression of the drug effect, although in what way is not clear.
ACKNOWLEDGEMENTS Although the research described in this article was funded in part by the United States EPA under assistance agreement number R809452 to the University of Arkansas for Medical Sciences, it has not been subjected to the Agency's peer and administrative review and, therefore, does not reflect the view of the Agency and no official endorsement should be inferred.
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7. Goldstein, A. Biostatistics: An Introductory Text. New York: MacMillan Co., 1964. 8. Gollub, L. R. The relations among measures of performance of fixed-interval schedules. J Exp Anal Behav 7: 337-343, 1964. 9. Hughes, J. A. and S. B. Sparber. d-Amphetamine unmasks postnatal consequences of exposure to methylmercury in utero: Methods for studying behavioral teratogenesis. Pharmacol Biochem Behav 9: 365-375, 1978. 10. Leander, J. D. and B. A. Gau. Flavor aversions rapidly produced by inorganic lead and triethyl tin. Neurotoxicology 1: 635-642, 1980. 11. McMillan, D. E. and J. D. Leander. Modification of baseline operant behavior by drugs. In: Behavioral Pharmacology, edited by S. D. Glick and J. Goldfarb. St Louis: Mosby Co., 1976, pp. 85-139. 12. McMillan, D. E. and A. T Miller. Interactions between carbon monoxide and d-amphetamine, or pentobarbital on schedulecontrolled behavior. Environ Res 8: 53-63, 1974. 13. McMillan, D. E. and G. R. Wenger. Neurobehavioral toxicology of trialkyltins. Pharrnacol Rev 37: 365-379, 1985.
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14. Organization for Economic Co-Operation and Development. Guidelines for the Testing of Chemicals. Paris, 1981. 15. Reiter, L. W., K. Kidd, G. Heavener and P. Rupert. Behavioral toxicity of acute and subacute exposure to triethyltin in the rat. Neurotoxicology 2: 97-112, 1980. 16. Squibb, R. E., N. G. Carmichael and H. A. Tilson. Behavioral and neuromorphological effects of triethyl tin bromide in adult rats. Toxicol Appl Pharmacol 55: 188-197, 1980. 17. Swartzwelder, H. S., R. S. Dyer, W. Holahan and R. D. Myers. Activity changes in rats following acute trimethyltin exposure. Neurotoxicology 2: 58%593, 1981.
McMILLAN ET AL. 18. U.S. Environmental Protection Agency. Proposed guidelines for registering pesticides in the United States: Hazard evaluation: Humans and domestic animals. Fed Reg 43: 37336-37403, 1978. 19. Walsh, T. J., J. M. Gallagher, E. Bostock and R. S. Dyer. Trimethyltin impairs retention of a passive avoidance task. Neurobehav Toxicol 4: 163-168, 1982. 20. Wenger, G. R., D. E. McMillan and L. W. Chang. Behavioral toxicology of acute trimethyltin exposure in the mouse. Neurobehav Toxicol Teratol 4: 157-161, 1982. 21. Wenger, G. R., D. E. McMillan and L. W. Chang. Behavioral effects of trimethyltin in two strains of mice, I1. Multiple fixed ratio, fixed interval. Toxicol Appl Pharmacol 73: 89-96, 1984.