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Behavioral and Monoaminergic Changes After Lindane Exposure in Developing Rats
Neurotoxicology and Teratology, Vol. 20, No. 2, pp. 155–160, 1998 © 1998 Elsevier Science Inc. Printed in the USA. All rights reserved 0892-0362/98 $19.00 1 .00
PII S0892-0362(97)00079-2
Behavioral and Monoaminergic Changes After Lindane Exposure in Developing Rats S. RIVERA,*1 R. ROSA,* E. MARTÍNEZ,† C. SUÑOL,† M. T. SERRANO,* M. VENDRELL,* E. RODRÍGUEZ-FARRÉ* AND C. SANFELIU* *Department of Pharmacology and Toxicology and †Department of Neurochemistry, IIBB, CSIC, c/Jordi Girona 18-26, E-08034 Barcelona, Spain Received 7 February 1997; Accepted 11 July 1997 RIVERA, S., R. ROSA, E. MARTÍNEZ, C. SUÑOL, M. T. SERRANO, M. VENDRELL, E. RODRÍGUEZ-FARRÉ AND C. SANFELIU. Behavioral and monoaminergic changes after lindane exposure in developing rats. NEUROTOXICOL TERATOL 20(2) 155–160, 1998.—The effects of lindane on behavior and central monoaminergic systems were studied in rat pups at 15 days of postnatal age. Pups were previously given nonconvulsant lindane PO doses, either a single 20 mg/kg or 7-day repeated 10 mg/kg doses. Both treatment schedules improved the passive avoidance acquisition but only the acute administration prolonged the step-through latency. Acute lindane decreased the motor activity, whereas the repeated dosing increased it. Increases of the ratio 5-HIAA/serotonin in several brain regions and of the ratio DOPAC/dopamine in the mesencephalon after a single dose of lindane suggest an enhanced monoaminergic turnover. In contrast, repeated lindane doses decreased monoamine/metabolite ratios excluding the striatum, where an increase of DOPAC/dopamine ratio correlates with the higher motor activity of these animals. It is postulated that both the imbalance of the central monoaminergic systems and the lindane-induced GABAergic blockade may be the basis of the behavioral alterations. © 1998 Elsevier Science Inc. Lindane g-Hexachlorocyclohexane Postnatal developing rat
Passive avoidance acquisition
THE chlorinated hydrocarbon lindane (g-hexachlorocyclohexane) is a crop insecticide and human and veterinary ectoparasiticide that has become an ubiquitous environmental contaminant. Lindane is a powerful central neurostimulant inducing convulsions and other signs of hyperexcitability in mammals [for review see (12,34)]. The effects of convulsant and subconvulsant doses of lindane have been extensively reported at different levels of biological organization in adult rats. For instance, lindane interferes with avoidance responding (38), modifies motor activity (16,18), produces anxiogenic effects (11,17), disturbs temperature and body weight regulation (4,43), facilitates kindling (9,13), and alters the neurochemical (2,24,37), metabolic (32), and blood flow (36) balance in the central nervous system. Several lines of evidence indicate that lower doses of lindane than those used in the aforementioned adult studies can also induce behavioral (27,28), neurochemical (30), metabolic (29), and even structural (33) changes in developing rats. Therefore, immature animals may be more vulnerable to the effects of lindane. On
Motor activity
Monoamines
the other hand, whether all these changes derive from the blockade of the GABAergic neurotransmission by lindane (1,8,22,26) or also reflect the interaction with other neurotransmitter systems is still an unsolved question. In this regard, minor changes of GABA regional concentrations but clear alterations of the monoaminergic status have been reported after convulsant lindane doses in adult rats (37). Few attempts to correlate monoamine alterations and behavioral changes in mature animals after lindane exposure have been carried out (15) and none at all during developmental stages. It was the aim of the present work to investigate if the modified behavioral profile in immature rats exposed to subconvulsant doses of lindane is related to the imbalance of the central monoaminergic systems. For this purpose, both a single and a 7-day repeated administration pattern were used because differential effects have been previously reported in adult rats and pups (29,32). Spontaneous motor activity and passive avoidance acquisition were selected as behaviors in developmental adjustment in the 2-week-old rat, which are
Requests for reprints should be addressed to Coral Sanfeliu, Department of Pharmacology and Toxicology, IIBB, CSIC, c/ Jordi Girona 1826, E-08034 Barcelona, Spain. Tel: 134-3-400 6141; Fax: 1 34-3-204 5904; E-mail: [email protected] 1 Present address: INSERM U. 29, Hôpital de Port Royal, 123 Bd. de Port Royal, 75014 Paris, France.
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very sensitive to any derangement of the immature central nervous system. In addition, regional brain concentration of the monoamines serotonin and dopamine and their respective acid metabolites 5-hydroxyindoleacetic acid (5-HIAA) and 3,4-dihydroxyphenylacetic acid (DOPAC) were analyzed. Metabolite/monoamine ratios were used as an index of neuronal activity. METHOD
Animals Pregnant Wistar rats were supply by Interfauna (St. Feliu de Codines, Barcelona, Spain). One day after birth all rat pups of each litter were randomly distributed among the different mothers to form experimental litters of 8––10 pups each, half males and half females. A total of eight experimental litters was used for the passive avoidance acquisition testing, six for the spontaneous motor activity testing, and six for the neurotransmitter determinations. In each set, experimental litters were randomly assigned to the different treatment groups described below. Offspring and foster mothers were housed under controlled conditions of 12-h light/dark cycle and 23˚C. Water and food were available ad lib. The research was conducted in compliance with the Spanish legislation on “Protection of Animals Used for Experimental and Other Scientific Purposes” and the European Communities Directive on this subject. Lindane Treatment Lindane was purchased from Merck (Darmstad, Germany) and dissolved in olive oil. Animals were intragastrically given a volume of 0.03 ml/10 g body weight using a Silastic® silicone tubing or small metallic cannulae and monitored for the presence of clinical signs. Four treatment groups were defined as follows: single administration of vehicle (I) or 20 mg/kg lindane (II) on day 15; repeated administration of vehicle (III) or 10 mg/kg lindane (IV) from postnatal day 8 to 14. Single 20 mg/kg lindane dosing did not induce convulsions in rat pups. The lower concentration of 10 mg/kg was used in the daily repeated treatment to avoid convulsions in these young animals because lindane accumulates in brain tissue (31). Testing times on day 15 at 1 h and 24 h after single and repeated administration, respectively, were chosen on the basis of our previous work with lindane in rat pups (27–30) and of lindane pharmacokinetics and accumulation in adult rats (31, 40). Spontaneous motor activity of groups III and IV was also recorded daily throughout the study. Two additional treatment groups were defined in the spontaneous motor activity study at single dose: daily vehicle administration from postnatal day 8 to 14 followed by a single administration of vehicle (V) or 20 mg/kg lindane (VI) on day 15. Results of treatments V and VI vs. treatments I and II, respectively, would show any effect of the repeated handling on the rat pups. Passive Avoidance Acquisition Testing Passive avoidance behavior was studied in a Plexiglas passive avoidance apparatus, with two separate compartments of equal dimensions (13 3 13 3 21 cm) that could be entered through a guillotine door. One compartment was lighted with a 15-W bulb and the other was dark. The grid floor of the dark compartment consisted of parallel steel bars (2 mm diameter) set 1 cm apart. The rat pup was initially placed in the lighted
compartment and the door was raised. The seconds the animal remained there until it entered the dark compartment were recorded as the latency time. After the rat entry, the guillotine door was lowered. The rat pup was returned to its cage for 1 min. Then the same procedure was performed again but an electric shock (0.5 mA 3 1 s) was delivered when the animal stepped onto the grid floor of the dark compartment with its four paws. The shock trial was repeated and the number of entries counted until the rat pup remained more than 1 min in the lighted part of the chamber. Retention of the passive avoidance task was not tested as these young animals showed a poor performance 24 h after acquisition. Spontaneous Motor Activity Recording Spontaneous motor activity was recorded with a videocomputerized system (Videotrack 512, View Point, Lyon, France) and measured by using a subtraction image analysis (10). In this method, each image is subtracted from the previous one to generate a shadow that was computed and analyzed in terms of quantity of movement. At the testing time, animals were individually placed in Polyglass cages (35.5 3 35.5 3 35.5 cm) in a soundproof room. Four cages, black to avoid visual contact between the animals during the test, were uniformly illuminated with two incandescent lamps (100 W) located 1.5 cm above the floor. A video-camera (CHU Inc., San Diego, CA) took an aerial view of the cages and sent the information to the video-computerized system in an adjoining room. Animal movement was analyzed and recorded for a 30-min session divided into 15 intervals of 2 min each. The mean seconds of motor activity per 2-min interval were calculated for each animal. Neurotransmitter Determinations Rat pups were decapitated and brains rapidly removed and dissected on a cold plate. Cerebral regions were stored at 280˚C until assayed for monoamine content. Serotonin, dopamine, and their respective acid metabolites 5-HIAA and DOPAC were simultaneously analyzed by an HPLC system (Waters, Milford, MA) with a C18 reversed phase column (Tracer Analítica, Barcelona) and electrochemical detection (Bioanalytical Systems, West Lafayette, IN) [as described in (15)]. Quantitative analyses were carried out by the external standard method. Serotonin and 5-HIAA were analyzed in eight regions: pons/medulla (PM), colliculi (COL), mesencephalon (MES), thalamus (T), hypothalamus (HT), hippocampus (HC), striatum (ST), frontal cortex (FC). Dopamine and DOPAC were analyzed in three regions: MES, HT, and ST. Results of the single-dose measurements were published in a previous article (30). Statistical Analysis Results were calculated as mean 6 SEM and evaluated with Student’s t-test or ANOVA where more than two groups of data were present. The data were analyzed jointly for both male and female offspring (50% each) because there were no statistical differences between male and female values within each treatment group. RESULTS
Clinical Signs Neither single nor repeated exposure to lindane at the doses tested produced seizures, tremors, weight loss, or other types of apparent behavioral or physiological dysfunctions.
LINDANE EFFECTS IN DEVELOPING RATS
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Passive Avoidance Acquisition
Spontaneous Motor Activity
Figure 1 shows the behavioral pattern in the passive avoidance paradigm at postnatal day 15 after single and repeated exposure to lindane. Single 20 mg/kg of lindane induced a statistically significant decrease of the number of entries and a significant increase of the latency time compared to the control group. On the other hand, the repeated treatment with 10 mg/kg of lindane also significantly reduced the number of entries but did not change the latency time.
Figure 2 shows that 20 mg/kg lindane single dose at postnatal day 15 caused a statistically significant decrease of the motor activity compared to the control group. The same treatment also induced a significant decrease in animals that were previously administered with olive oil from day 8 to day 14. Figure 3 shows the results of the repeated exposure to 10 mg/ kg of lindane. Testing was performed along the whole administration period, 24 h after each dosing. This treatment induced the opposite tendency, namely, a significant increase of the motor activity on lindane-dosed animals compared to controls. Neurotransmitter Ratios Figures 4 and 5 show the ratios of 5-HIAA/serotonin and DOPAC/dopamine, respectively. The ratio 5-HIAA/serotonin showed a generalized increase after a single dose of 20 mg/ kg of lindane compared to control. Increases were statistically significant in the hippocampus and the frontal cortex. In contrast, the repeated exposure to 10 mg/kg of the neurotoxicant caused decreases that were statistically significant in the pons medulla, the colliculi, and the frontal cortex. However, the striatum showed a tendency to increase the ratio after both treatments. The ratio DOPAC/dopamine tended to increase in all the studied areas after a single dose of lindane, being statistically significant in the mesencephalon. After the repeated treatment with lindane, a significant decrease was found in the mesencephalon whereas a marked increase took place in the striatum. DISCUSSION
The present results, as summarized in Table 1, provide evidence that nonconvulsant lindane doses alter the behavior and the central monoaminergic balance of developing rats. The single-dose treatment of lindane improved the acquisition of the passive avoidance task. Even though this is a surprising effect for such a neurotoxic agent, it would be in agreement with the lindane effects on long-term potentiation
FIG. 1. Acquisition of passive avoidance in rat pups on the postnatal day 15: 1 h after a 20 mg/kg lindane dose (top) and 24 h after a 7-day repeated dosing of 10 mg/kg lindane (bottom). Left Y axis: number of entries in the dark compartment until the animal remained for more than 1 min in the lighted compartment. Right Y axis: latency time in seconds until the animal entered for the first time (preshock) into the dark compartment. Values represent the mean 6 SEM of 16–18 animals. **p , 0.01 (Student’s t-test) compared to control.
FIG. 2. Spontaneous motor activity of 15-day-old rats recorded 1 h after a single dose of lindane 20 mg/kg: animals not previously handled (naive, left histogram) and animals administrated with the vehicle (olive oil) from postnatal day 8 to 14 (nonnaive, right histogram). Motor activity is expressed in seconds of movement per 2-min interval. Values represent the mean 6 SEM of 8 animals. *p , 0.05 (Student’s t-test) compared to control.
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FIG. 3. Spontaneous motor activity curve of rat pups along postnatal days 9 to 15 recorded 24 h after each 7-day repeated dose of 10 mg/kg lindane. Values represent the mean 6 SEM of 7–8 animals. Motor activity is expressed in seconds of movement per 2-min interval. Curve statistically significant [repeated-measures ANOVA: F(1, 29) 5 4.99, p 5 0.03] compared to control curve.
in the hippocampus (42,43). Agents other than lindane that block chloride-induced GABAergic currents, like picrotoxinin, have been reported to enhance retention of an inhibitory avoidance response (3,5). Both compounds seem to have the same binding site on the GABAA receptor–ionophore complex (1,20,25,35). However, single-dose studies in adult rats have shown that lindane impairs retention of aversive avoidance tasks, also attributed to a GABAergic alteration (38). On the other hand, 20 mg/kg of lindane caused a marked reduction of the spontaneous motor activity. A lindanedepressed animal activity can cause the step-through latency increase in the passive avoidance test. It is not clear if these effects may be related to the anxiogenic effect of lindane previously reported in rat pups (27). Antagonists of GABAA receptor complex, like picrotoxinin and pentylenetetrazol, also display anxiogenic effects in some animal models of anxiety (39). A high anxiety state correlates positively with an increased serotonergic activity (7,21) and, interestingly, the present results showed highest increases of serotonin turnover in limbic system-related structures like the hippocampus or the frontal cortex. The increase of dopamine metabolism in the mesencephalon, where the main dopaminergic cell bodies are located, indicates that this neurotransmitter could also be involved in the aforementioned changes of the developmental behavioral profile. Anxiogenic effects are also present in adults (11,17). Besides, no decreases of motor activity but pattern changes and increases have been reported in adult rats (16,18). Serotonergic and dopaminergic neurons have also been found activated in mature rats (2) and at different postnatal ages (30) after a subconvulsant lindane dose. The repeated dose with lindane improved the acquisition of the passive avoidance behavior similar to the single treat-
FIG. 4. 5-HIAA/serotonin ratio in different rat brain regions on postnatal day 15: 1 h after a 20 mg/kg single dose of lindane or 24 h after a 7-day repeated dosing of 10 mg/kg. Values represent the mean 6 SEM of 7–9 animals, normalized by control values (100%). *p , 0.05, **p , 0.01 (Student’s t-test) compared to control. Abbreviations: PM, pons/medulla; COL, colliculi; MES, mesencephalon; T, thalamus; HT, hypothalamus; HC, hippocampus; ST, striatum; FC, frontal cortex. Regional concentrations (ng/g tissue) of metabolite/ neurotransmitter in the single dosed control group were: 306 6 19/270 6 12 (PM); 419 6 28/422 6 31 (COL); 461 6 38/329 6 13 (MES); 208 6 15/93 6 9 (T); 426 6 34/432 6 22 (HT); 84 6 5/73 6 4 (HC); 150 6 13/94 6 9 (ST); 177 6 9/283 6 16 (FC). Concentrations (ng/g tissue) in the repeatedly dosed control animals were: 371 6 15/316 6 8 (PM); 334 6 20 /430 6 32 (COL); 501 6 25/377 6 36 (MES); 244 6 10/136 6 9 (T); 302 6 14/268 6 16 (HT); 97 6 4/86 6 4 (HC); 171 6 9/128 6 11 (ST); 193 6 27/287 6 42 (FC).
LINDANE EFFECTS IN DEVELOPING RATS
159 TABLE 1 SUMMARY OF THE EFFECTS OBSERVED IN 15-DAY-OLD RATS 1 h AFTER A SINGLE DOSE OF 20 mg/kg OR 24 h AFTER 7-DAY REPEATED DOSES OF 10 mg/kg LINDANE Lindane Treatment Single
Passive avoidance acquisition Latency time in passive avoidance Spontaneous motor activity Ratio 5-HIAA/serotonin Ratio DOPAC/dopamine
Repeated
Increased Increased
Increased Unchanged
Reduced Increased (HC, FC) Increased (MES)
Increased Decreased (PM, COL, FC) Decreased (MES)/ increased (ST)
See Figs. 1–5 for actual values. See Fig. 4 for abbreviations.
FIG. 5. DOPAC/dopamine ratio in different rat brain regions on postnatal day 15: 1 h after a 20 mg/kg single dose of lindane or 24 h after a 7-day repeated dosing of 10 mg/kg. Values represent the mean 6 SEM of 7–9 animals, normalized by control values (100%). *p , 0.05 (Student’s t-test) compared to control. For abbreviations see Fig. 4. Regional concentrations (ng/g tissue) of metabolite/neurotransmitter in the single dosed control group were: 131 6 17/293 6 16 (MES); 612 6 57/2594 6 126 (ST); 59 6 6/201 6 18 (HT). Concentrations (ng/g tissue) in the repeatedly dosed control animals were: 34 6 2/190 6 12 (MES); 614 6 32/3043 6 180 (ST); 30 6 2/143 6 10 (HT).
ment. The latency time was not altered in the repeated treatment, even though the spontaneous motor activity appeared increased. This suggests that the level of activity did not interfere with the cognitive function. It has been hypothesized that an increased nigrostriatal dopamine metabolism would be in accordance with an increased motor activity (41). In support of the hypothesis would be the increase of DOPAC/dopamine ratio found in the striatum, along with a decrease in the mesencephalon. The system would respond by favoring the release and metabolization of dopamine in the striatum (area of projection), which plays a key role in the control of motor activity. At the same time this would be enhanced by reducing dopamine metabolism in the mesencephalon, where the amine is mainly synthesized, and thus increasing the availability of neurotransmitter in the striatum terminals. On the other hand, serotonin metabolism displayed a general tendency to decrease after the repeated exposure to the neurotoxicant, both in areas of synthesis (pons medulla) and projection (frontal cortex or colliculi). Only the striatum showed a slight nonsignificant increase of the 5-HIAA/serotonin ratio. The differential monoamine status after single and repeated doses may be preferentially linked to the differential motor activity changes. As regards to the anxiogenic effects, rat pups dosed
with 10 mg/kg lindane during the second postnatal week show an increase of ultrasonic vocalizations as do animals dosed once with 20 mg/kg lindane (27). It is tempting to hypothesize that the lindane-induced improvement of the passive avoidance acquisition and the anxiogenic effects, which appear after both single and repeated treatment, are mainly caused by GABAergic disturbances. The immaturity of the rat brain during the second postnatal week, particularly the serotonergic system (14,19), could account for the different response to lindane exposure compared to adults. In addition, the lindane impairment of the myelinization process of the rat throughout the second postnatal week (33) may contribute to the monoamine and behavior profile exhibited by young animals, depending on the age and the treatment schedule. Also in developing rats, a peak of hyperactivity between postnatal days 12–16 has been described after repeated exposure to lindane (28). In adult rats, repeated small doses of lindane have been reported to impair learning capabilities (6) whereas convulsant ones decrease dopamine levels (24). Dieldrin, a related organochloride insecticide, was reported to enhance learning in the adult rat when administrated at very low doses beginning on day 5 of gestation (23). In summary, the present work shows that subconvulsant doses of lindane elicit neurochemical and behavioral effects in developing rats that may be different from those reported in adults, and partially depending on the treatment schedule. It can be concluded that there is some correlation between neurochemical and behavioral data after lindane exposure. Imbalance of both GABA and monoaminergic systems may be the basis of the behavioral disturbances observed in the immature rat. Monoamine alterations may cause the differential acute vs. repeated dosing behavior disturbances, whereas changes of GABAergic function perhaps contribute to the improved passive avoidance retention. Finally, the results support the idea that the limbic and motor systems are major targets for lindane in the central nervous system. ACKNOWLEDGEMENTS
This work was supported by research grants SAF94-0076 and SAF96-0129 from the Spanish Comisión Interministerial de Ciencia y Tecnología (CICYT). The authors gratefully acknowledge the expert technical assistance of C. Cleries and E. Bustamante.
REFERENCES 1. Abalis, I. M.; Eldefrawi, M. E.; Eldefrawi, A. T.: High-affinity binding of cyclodiene insecticides and g-hexachlorocyclohexane
to g-aminobutyric acid receptors of rat brain. Pestic. Biochem. Physiol. 24:95–102; 1985.
160 2. Artigas, F.; Martínez, E.; Camón, L.; Rodríguez-Farré, E.: Synthesis and utilization of neurotransmitters: Actions of subconvulsant doses of hexachlorocyclohexane isomers on brain monoamines. Toxicology 49:49–55; 1988. 3. Brioni, J. D.; McGaugh, J. L.: Posttraining administration of GABAergic antagonists enhances retention of aversively motivated tasks. Psychopharmacology (Berlin) 96:505–510; 1985. 4. Camón, L.; Martínez, E; Artigas, F.; Solà, C.; Rodríguez-Farré E.: The effect of nonconvulsant doses of lindane on temperature and body weight. Toxicology 49:389–394; 1988. 5. Castellano, C.; Introini-Collison, I. B.; McGaugh, J. L.: Interaction of beta-endorphin and GABAergic drugs in the regulation of memory storage. Behav. Neural Biol. 60:123–128; 1993. 6. Dési, I.: Neurotoxicological effects of small quantities of lindane. Int. Arch. Arbeitsmed. 33:153–162; 1974. 7. Dourish, C. T.; Hutson, P. H.; Curzon, G.: Putative anxiolytic 8-OH-DPAT, buspirone and TVX Q 7821 are agonist at 5-HT1A autoreceptors in the raphé nuclei. Trends Pharmacol. Sci. 17:212– 214; 1986. 8. Ghiasuddin, S. M.; Matsumura, F.: Inhibition of g-aminobutyric acid (GABA)-induced chloride uptake by g-BHC and heptachlorepoxide. Comp. Biochem. Physiol. [C] 73:141–144; 1982. 9. Gilbert, M. E.: Repeated exposure to lindane leads to behavioral sensitization and facilitates electrical kindling. Neurotoxicol. Teratol. 17:131–141; 1995. 10. Giménez-Llort, L.; Martínez, E; Ferré, S.: Dopamine-independent and adenosine-dependent mechanisms involved in the effects of N-methyl-D-aspartate on motor activity in mice. Eur. J. Pharmacol. 275:171–177; 1995. 11. Hijzen, T. H.; Slagen, J. L.: Effects of midazolam, DMCM and lindane on potentiated startle in the rat. Psychopharmacology (Berlin) 99:362–365; 1989. 12. Joy, R. M.: Chlorinated hydrocarbon insecticides. In: Ecobichon, D. L.; Joy, R. M., eds. Pesticides and neurological diseases. Boca Raton: CRC Press; 1994:81–170. 13. Joy, R. M.; Stark, L. G.; Albertson, T. E.: Proconvulsant effects of lindane: Enhancement of amygdaloid kindling in the rat. Neurobehav. Toxicol. Teratol. 4:347–354; 1982. 14. Lidov, H. G. W.; Milliver, M E.: An immunohistochemical study of serotonin neurons development in the rat: Ascending pathways and terminal fields. Brain Res. Bull. 8:389–430; 1982. 15. Llorens, J.; Suñol, C.; Tusell, J. M.; Rodríguez-Farré, E.: Evidence for acute tolerance to the behavioral effects of lindane: Concomitant changes in regional monoamine status. Neurotoxicology 12:697–706; 1991. 16. Llorens, J.; Tusell, J. M.; Suñol, C.; Rodríguez-Farré, E.: Effects of lindane on spontaneous behavior of rats analyzed by multivariate statistics. Neurotoxicol. Teratol. 11:145–151; 1989. 17. Llorens, J.; Tusell, J. M.; Suñol, C.; Rodríguez-Farré, E.: On the effects of lindane on the plus maze model of anxiety. Neurotoxicol. Teratol. 12:643–647; 1990. 18. Llorens, J.; Tusell, J. M.; Suñol, C.; Rodríguez-Farré, E.: Repeated lindane exposure in the rat results in changes in spontaneous motor activity at 2 weeks postexposure. Toxicol. Lett. 61:265–274; 1992. 19. Marbry, P. D.; Campbell, B. A.: Ontogeny of serotonergic inhibition of behavioral arousal in the rat. J. Comp. Physiol. Psychol. 86:193–201; 1974. 20. Matsumura, F.; Ghiasuddin, S. M.: Evidence for similarities between cyclodiene type insecticides and picrotoxin in their action mechanisms. J. Environ. Sci. Health B18:1–14; 1983. 21. Moser, P. C.; Tricklebank, M. D.; Middlemiss, D. N.; Mir, A. K.; Hibert, M. F.; Fozar, J. R.: Characterization of MDL 73005EF as a 5-HT1A selective ligand and its effects in animal models of anxiety: Comparison with buspirone, 8-OH-DPAT and diazepam. Br. J. Pharmacol. 99:343–349; 1990. 22. Obata, T.; Yamamura, H. I.; Malatynska, E.; Ikeda, M.; Laird, H.; Palmer, C. J.; Casida, J. E.: Modulation of gamma-aminobutyricstimulated chloride influx by bicycloorthocarboxylates, bycyclophosphorus esters, polychlorocycloalkanes and other cage convulsants. J. Pharmacol. Exp. Ther. 244:802–806; 1988. 23. Olson, K. L.; Bousch, G. M.; Matsumura, F.: Pre- and postnatal
RIVERA ET AL.
24. 25. 26.
27. 28. 29.
30.
31. 32.
33. 34. 35.
36. 37.
38. 39. 40.
41.
42. 43.
exposure to dieldrin: Persistent stimulatory and behavioral effects. Pestic. Biochem. Physiol. 13:20–33; 1980. Ortiz-Martínez, A.; Martínez-Conde, E.: The neurotoxic effects of lindane at acute and subchronic dosages. Ecotoxicol. Environ. Safety 30:101–105; 1995. Pomés, A.; Rodríguez-Farré, E.; Suñol, C.: Inhibition of t-butylbicyclophosphorothionate binding by convulsant agents in primary cultures of cerebellar neurons. Dev. Brain Res. 73:85–90; 1993. Pomés, A.; Rodríguez-Farré, E.; Suñol, C.: Disruption of GABAdependent chloride flux by cyclodienes and hexachlorocyclohexanes in primary cultures of cortical neurons. J. Pharmacol. Exp. Ther. 271:1616–1623; 1994. Rivera, S.; Sanfeliu, C.; García, F.; Comellas, F.; Rodríguez-Farré E.: Increase of rat pup ultrasonic isolation calls induced by lindane. Neurotoxicology 13:235–240; 1992. Rivera, S.; Sanfeliu, C.; Rodríguez-Farré, E.: Behavioral changes induced in developing rats by an early postnatal exposure to lindane. Neurotoxicol. Teratol. 12:591–595; 1990. Rivera, S.; Sanfeliu, C.; Rodríguez-Farré, E.: Changes in regional brain 2-14C-deoxyglucose uptake induced in postnatal developing rats by single and repeated nonconvulsant doses of lindane. Pestic. Biochem. Physiol. 43:241–252; 1992. Rivera, S.; Sanfeliu, C.; Suñol, C.; Rodríguez-Farré, E.: Regional effects on the cerebral concentration of noradrenaline, serotonin and dopamine in suckling rats after a single dose of lindane. Toxicology 69:43–54; 1991. Sanfeliu, C.; Camón, L.; Martínez, E.; Solà, C.; Artigas, F.; Rodríguez-Farré, E.: Regional distribution of lindane in rat brain. Toxicology 49:189–196; 1988. Sanfeliu, C.; Solà, C.; Camón, L.; Martínez, E.; Rodríguez-Farré, E.: Regional changes in brain 2-14C-deoxyglucose uptake induced by convulsant and nonconvulsant doses of lindane. Neurotoxicology 10:727–742; 1989. Serrano, M. T.; Vendrell, M.; Rivera, S.; Serratosa, J.; RodríguezFarré, E.: Effects of lindane on the myelination process in the rat. Neurotoxicol. Teratol. 12:577–583; 1990. Smith, A. G.: Chlorinated hydrocarbon insecticides. In: Hayes, W. J.; Laws, E. R., eds. Handbook of pesticide toxicology. San Diego: Academic Press; 1991:731–915. Solà, C.; Martínez, E.; Camón, L.; Pazos, A.; Rodríguez-Farré, E.: Lindane administration to the rat induces modifications in the regional cerebral binding of [3H]muscimol, [3H]flunitrazepam, and t-[3S]butylbicyclophosphorothionate: An autoradiographic study. J. Neurochem. 60:1821–1834; 1993. Solà, C.; Planas, A.M.; Camón, L.; Martínez, E; Rodríguez-Farré E.: Study of regional cerebral blood flow after lindane administration to the rat. Pestic. Biochem. Physiol. 38:1–8; 1990. Suñol, C.; Tusell, J. M.; Gelpí, E.; Rodríguez-Farré, E.: Regional concentrations of GABA, and noradrenaline in brain at onset of seizures induced by lindane (gamma-hexachlorocyclohexane). Neuropharmacology 27:677–681; 1988. Tilson, H. A.; Shaw, S.; McLamb, R. L.: The effects of lindane, DDT, and chlordecone on avoidance responding and seizure activity. Toxicol. Appl. Pharmacol. 88:57–65; 1987. Treit, D.: Ro 15-1788, CGS 8216, picrotoxin, and pentylenetetrazol: Do they antagonize anxiolytic drug effects through an anxiogenic action? Brain Res. Bull. 19:401–405; 1987. Tusell, J. M.; Suñol, C.; Gelpí, E.; Rodríguez-Farré, E.: Relationship between lindane concentration in blood and brain and convulsant response in rats after oral or intraperitoneal administration. Arch. Toxicol. 60:432–437; 1987. van Praag, H. M.; Asnis, G. M.; Kahn, R. S.; Brown, S. L.; Korn, M.; Friedman, J. M.; Wetzler, S.: Monoamines and abnormal behaviour. A multi-aminergic perspective. Br. J. Psychiatry 157: 723–734; 1990. Woolley, D.; Zimmer, L.; Dodge, D.; Swanson, K.: Effects of lindane-type insecticides in mammals: Unsolved problems. Neurotoxicology 6:165–192; 1985. Woolley, D.; Zimmer, L.; Hasan, Z.; Swanson, K.: Do some insecticides and heavy metals produce long-term potentiation in the limbic system? In: Narahashi, T., ed. Cellular and molecular neurotoxicology. New York: Raven Press; 1984:45–69.
PII S0892-0362(97)00079-2
Behavioral and Monoaminergic Changes After Lindane Exposure in Developing Rats S. RIVERA,*1 R. ROSA,* E. MARTÍNEZ,† C. SUÑOL,† M. T. SERRANO,* M. VENDRELL,* E. RODRÍGUEZ-FARRÉ* AND C. SANFELIU* *Department of Pharmacology and Toxicology and †Department of Neurochemistry, IIBB, CSIC, c/Jordi Girona 18-26, E-08034 Barcelona, Spain Received 7 February 1997; Accepted 11 July 1997 RIVERA, S., R. ROSA, E. MARTÍNEZ, C. SUÑOL, M. T. SERRANO, M. VENDRELL, E. RODRÍGUEZ-FARRÉ AND C. SANFELIU. Behavioral and monoaminergic changes after lindane exposure in developing rats. NEUROTOXICOL TERATOL 20(2) 155–160, 1998.—The effects of lindane on behavior and central monoaminergic systems were studied in rat pups at 15 days of postnatal age. Pups were previously given nonconvulsant lindane PO doses, either a single 20 mg/kg or 7-day repeated 10 mg/kg doses. Both treatment schedules improved the passive avoidance acquisition but only the acute administration prolonged the step-through latency. Acute lindane decreased the motor activity, whereas the repeated dosing increased it. Increases of the ratio 5-HIAA/serotonin in several brain regions and of the ratio DOPAC/dopamine in the mesencephalon after a single dose of lindane suggest an enhanced monoaminergic turnover. In contrast, repeated lindane doses decreased monoamine/metabolite ratios excluding the striatum, where an increase of DOPAC/dopamine ratio correlates with the higher motor activity of these animals. It is postulated that both the imbalance of the central monoaminergic systems and the lindane-induced GABAergic blockade may be the basis of the behavioral alterations. © 1998 Elsevier Science Inc. Lindane g-Hexachlorocyclohexane Postnatal developing rat
Passive avoidance acquisition
THE chlorinated hydrocarbon lindane (g-hexachlorocyclohexane) is a crop insecticide and human and veterinary ectoparasiticide that has become an ubiquitous environmental contaminant. Lindane is a powerful central neurostimulant inducing convulsions and other signs of hyperexcitability in mammals [for review see (12,34)]. The effects of convulsant and subconvulsant doses of lindane have been extensively reported at different levels of biological organization in adult rats. For instance, lindane interferes with avoidance responding (38), modifies motor activity (16,18), produces anxiogenic effects (11,17), disturbs temperature and body weight regulation (4,43), facilitates kindling (9,13), and alters the neurochemical (2,24,37), metabolic (32), and blood flow (36) balance in the central nervous system. Several lines of evidence indicate that lower doses of lindane than those used in the aforementioned adult studies can also induce behavioral (27,28), neurochemical (30), metabolic (29), and even structural (33) changes in developing rats. Therefore, immature animals may be more vulnerable to the effects of lindane. On
Motor activity
Monoamines
the other hand, whether all these changes derive from the blockade of the GABAergic neurotransmission by lindane (1,8,22,26) or also reflect the interaction with other neurotransmitter systems is still an unsolved question. In this regard, minor changes of GABA regional concentrations but clear alterations of the monoaminergic status have been reported after convulsant lindane doses in adult rats (37). Few attempts to correlate monoamine alterations and behavioral changes in mature animals after lindane exposure have been carried out (15) and none at all during developmental stages. It was the aim of the present work to investigate if the modified behavioral profile in immature rats exposed to subconvulsant doses of lindane is related to the imbalance of the central monoaminergic systems. For this purpose, both a single and a 7-day repeated administration pattern were used because differential effects have been previously reported in adult rats and pups (29,32). Spontaneous motor activity and passive avoidance acquisition were selected as behaviors in developmental adjustment in the 2-week-old rat, which are
Requests for reprints should be addressed to Coral Sanfeliu, Department of Pharmacology and Toxicology, IIBB, CSIC, c/ Jordi Girona 1826, E-08034 Barcelona, Spain. Tel: 134-3-400 6141; Fax: 1 34-3-204 5904; E-mail: [email protected] 1 Present address: INSERM U. 29, Hôpital de Port Royal, 123 Bd. de Port Royal, 75014 Paris, France.
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very sensitive to any derangement of the immature central nervous system. In addition, regional brain concentration of the monoamines serotonin and dopamine and their respective acid metabolites 5-hydroxyindoleacetic acid (5-HIAA) and 3,4-dihydroxyphenylacetic acid (DOPAC) were analyzed. Metabolite/monoamine ratios were used as an index of neuronal activity. METHOD
Animals Pregnant Wistar rats were supply by Interfauna (St. Feliu de Codines, Barcelona, Spain). One day after birth all rat pups of each litter were randomly distributed among the different mothers to form experimental litters of 8––10 pups each, half males and half females. A total of eight experimental litters was used for the passive avoidance acquisition testing, six for the spontaneous motor activity testing, and six for the neurotransmitter determinations. In each set, experimental litters were randomly assigned to the different treatment groups described below. Offspring and foster mothers were housed under controlled conditions of 12-h light/dark cycle and 23˚C. Water and food were available ad lib. The research was conducted in compliance with the Spanish legislation on “Protection of Animals Used for Experimental and Other Scientific Purposes” and the European Communities Directive on this subject. Lindane Treatment Lindane was purchased from Merck (Darmstad, Germany) and dissolved in olive oil. Animals were intragastrically given a volume of 0.03 ml/10 g body weight using a Silastic® silicone tubing or small metallic cannulae and monitored for the presence of clinical signs. Four treatment groups were defined as follows: single administration of vehicle (I) or 20 mg/kg lindane (II) on day 15; repeated administration of vehicle (III) or 10 mg/kg lindane (IV) from postnatal day 8 to 14. Single 20 mg/kg lindane dosing did not induce convulsions in rat pups. The lower concentration of 10 mg/kg was used in the daily repeated treatment to avoid convulsions in these young animals because lindane accumulates in brain tissue (31). Testing times on day 15 at 1 h and 24 h after single and repeated administration, respectively, were chosen on the basis of our previous work with lindane in rat pups (27–30) and of lindane pharmacokinetics and accumulation in adult rats (31, 40). Spontaneous motor activity of groups III and IV was also recorded daily throughout the study. Two additional treatment groups were defined in the spontaneous motor activity study at single dose: daily vehicle administration from postnatal day 8 to 14 followed by a single administration of vehicle (V) or 20 mg/kg lindane (VI) on day 15. Results of treatments V and VI vs. treatments I and II, respectively, would show any effect of the repeated handling on the rat pups. Passive Avoidance Acquisition Testing Passive avoidance behavior was studied in a Plexiglas passive avoidance apparatus, with two separate compartments of equal dimensions (13 3 13 3 21 cm) that could be entered through a guillotine door. One compartment was lighted with a 15-W bulb and the other was dark. The grid floor of the dark compartment consisted of parallel steel bars (2 mm diameter) set 1 cm apart. The rat pup was initially placed in the lighted
compartment and the door was raised. The seconds the animal remained there until it entered the dark compartment were recorded as the latency time. After the rat entry, the guillotine door was lowered. The rat pup was returned to its cage for 1 min. Then the same procedure was performed again but an electric shock (0.5 mA 3 1 s) was delivered when the animal stepped onto the grid floor of the dark compartment with its four paws. The shock trial was repeated and the number of entries counted until the rat pup remained more than 1 min in the lighted part of the chamber. Retention of the passive avoidance task was not tested as these young animals showed a poor performance 24 h after acquisition. Spontaneous Motor Activity Recording Spontaneous motor activity was recorded with a videocomputerized system (Videotrack 512, View Point, Lyon, France) and measured by using a subtraction image analysis (10). In this method, each image is subtracted from the previous one to generate a shadow that was computed and analyzed in terms of quantity of movement. At the testing time, animals were individually placed in Polyglass cages (35.5 3 35.5 3 35.5 cm) in a soundproof room. Four cages, black to avoid visual contact between the animals during the test, were uniformly illuminated with two incandescent lamps (100 W) located 1.5 cm above the floor. A video-camera (CHU Inc., San Diego, CA) took an aerial view of the cages and sent the information to the video-computerized system in an adjoining room. Animal movement was analyzed and recorded for a 30-min session divided into 15 intervals of 2 min each. The mean seconds of motor activity per 2-min interval were calculated for each animal. Neurotransmitter Determinations Rat pups were decapitated and brains rapidly removed and dissected on a cold plate. Cerebral regions were stored at 280˚C until assayed for monoamine content. Serotonin, dopamine, and their respective acid metabolites 5-HIAA and DOPAC were simultaneously analyzed by an HPLC system (Waters, Milford, MA) with a C18 reversed phase column (Tracer Analítica, Barcelona) and electrochemical detection (Bioanalytical Systems, West Lafayette, IN) [as described in (15)]. Quantitative analyses were carried out by the external standard method. Serotonin and 5-HIAA were analyzed in eight regions: pons/medulla (PM), colliculi (COL), mesencephalon (MES), thalamus (T), hypothalamus (HT), hippocampus (HC), striatum (ST), frontal cortex (FC). Dopamine and DOPAC were analyzed in three regions: MES, HT, and ST. Results of the single-dose measurements were published in a previous article (30). Statistical Analysis Results were calculated as mean 6 SEM and evaluated with Student’s t-test or ANOVA where more than two groups of data were present. The data were analyzed jointly for both male and female offspring (50% each) because there were no statistical differences between male and female values within each treatment group. RESULTS
Clinical Signs Neither single nor repeated exposure to lindane at the doses tested produced seizures, tremors, weight loss, or other types of apparent behavioral or physiological dysfunctions.
LINDANE EFFECTS IN DEVELOPING RATS
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Passive Avoidance Acquisition
Spontaneous Motor Activity
Figure 1 shows the behavioral pattern in the passive avoidance paradigm at postnatal day 15 after single and repeated exposure to lindane. Single 20 mg/kg of lindane induced a statistically significant decrease of the number of entries and a significant increase of the latency time compared to the control group. On the other hand, the repeated treatment with 10 mg/kg of lindane also significantly reduced the number of entries but did not change the latency time.
Figure 2 shows that 20 mg/kg lindane single dose at postnatal day 15 caused a statistically significant decrease of the motor activity compared to the control group. The same treatment also induced a significant decrease in animals that were previously administered with olive oil from day 8 to day 14. Figure 3 shows the results of the repeated exposure to 10 mg/ kg of lindane. Testing was performed along the whole administration period, 24 h after each dosing. This treatment induced the opposite tendency, namely, a significant increase of the motor activity on lindane-dosed animals compared to controls. Neurotransmitter Ratios Figures 4 and 5 show the ratios of 5-HIAA/serotonin and DOPAC/dopamine, respectively. The ratio 5-HIAA/serotonin showed a generalized increase after a single dose of 20 mg/ kg of lindane compared to control. Increases were statistically significant in the hippocampus and the frontal cortex. In contrast, the repeated exposure to 10 mg/kg of the neurotoxicant caused decreases that were statistically significant in the pons medulla, the colliculi, and the frontal cortex. However, the striatum showed a tendency to increase the ratio after both treatments. The ratio DOPAC/dopamine tended to increase in all the studied areas after a single dose of lindane, being statistically significant in the mesencephalon. After the repeated treatment with lindane, a significant decrease was found in the mesencephalon whereas a marked increase took place in the striatum. DISCUSSION
The present results, as summarized in Table 1, provide evidence that nonconvulsant lindane doses alter the behavior and the central monoaminergic balance of developing rats. The single-dose treatment of lindane improved the acquisition of the passive avoidance task. Even though this is a surprising effect for such a neurotoxic agent, it would be in agreement with the lindane effects on long-term potentiation
FIG. 1. Acquisition of passive avoidance in rat pups on the postnatal day 15: 1 h after a 20 mg/kg lindane dose (top) and 24 h after a 7-day repeated dosing of 10 mg/kg lindane (bottom). Left Y axis: number of entries in the dark compartment until the animal remained for more than 1 min in the lighted compartment. Right Y axis: latency time in seconds until the animal entered for the first time (preshock) into the dark compartment. Values represent the mean 6 SEM of 16–18 animals. **p , 0.01 (Student’s t-test) compared to control.
FIG. 2. Spontaneous motor activity of 15-day-old rats recorded 1 h after a single dose of lindane 20 mg/kg: animals not previously handled (naive, left histogram) and animals administrated with the vehicle (olive oil) from postnatal day 8 to 14 (nonnaive, right histogram). Motor activity is expressed in seconds of movement per 2-min interval. Values represent the mean 6 SEM of 8 animals. *p , 0.05 (Student’s t-test) compared to control.
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FIG. 3. Spontaneous motor activity curve of rat pups along postnatal days 9 to 15 recorded 24 h after each 7-day repeated dose of 10 mg/kg lindane. Values represent the mean 6 SEM of 7–8 animals. Motor activity is expressed in seconds of movement per 2-min interval. Curve statistically significant [repeated-measures ANOVA: F(1, 29) 5 4.99, p 5 0.03] compared to control curve.
in the hippocampus (42,43). Agents other than lindane that block chloride-induced GABAergic currents, like picrotoxinin, have been reported to enhance retention of an inhibitory avoidance response (3,5). Both compounds seem to have the same binding site on the GABAA receptor–ionophore complex (1,20,25,35). However, single-dose studies in adult rats have shown that lindane impairs retention of aversive avoidance tasks, also attributed to a GABAergic alteration (38). On the other hand, 20 mg/kg of lindane caused a marked reduction of the spontaneous motor activity. A lindanedepressed animal activity can cause the step-through latency increase in the passive avoidance test. It is not clear if these effects may be related to the anxiogenic effect of lindane previously reported in rat pups (27). Antagonists of GABAA receptor complex, like picrotoxinin and pentylenetetrazol, also display anxiogenic effects in some animal models of anxiety (39). A high anxiety state correlates positively with an increased serotonergic activity (7,21) and, interestingly, the present results showed highest increases of serotonin turnover in limbic system-related structures like the hippocampus or the frontal cortex. The increase of dopamine metabolism in the mesencephalon, where the main dopaminergic cell bodies are located, indicates that this neurotransmitter could also be involved in the aforementioned changes of the developmental behavioral profile. Anxiogenic effects are also present in adults (11,17). Besides, no decreases of motor activity but pattern changes and increases have been reported in adult rats (16,18). Serotonergic and dopaminergic neurons have also been found activated in mature rats (2) and at different postnatal ages (30) after a subconvulsant lindane dose. The repeated dose with lindane improved the acquisition of the passive avoidance behavior similar to the single treat-
FIG. 4. 5-HIAA/serotonin ratio in different rat brain regions on postnatal day 15: 1 h after a 20 mg/kg single dose of lindane or 24 h after a 7-day repeated dosing of 10 mg/kg. Values represent the mean 6 SEM of 7–9 animals, normalized by control values (100%). *p , 0.05, **p , 0.01 (Student’s t-test) compared to control. Abbreviations: PM, pons/medulla; COL, colliculi; MES, mesencephalon; T, thalamus; HT, hypothalamus; HC, hippocampus; ST, striatum; FC, frontal cortex. Regional concentrations (ng/g tissue) of metabolite/ neurotransmitter in the single dosed control group were: 306 6 19/270 6 12 (PM); 419 6 28/422 6 31 (COL); 461 6 38/329 6 13 (MES); 208 6 15/93 6 9 (T); 426 6 34/432 6 22 (HT); 84 6 5/73 6 4 (HC); 150 6 13/94 6 9 (ST); 177 6 9/283 6 16 (FC). Concentrations (ng/g tissue) in the repeatedly dosed control animals were: 371 6 15/316 6 8 (PM); 334 6 20 /430 6 32 (COL); 501 6 25/377 6 36 (MES); 244 6 10/136 6 9 (T); 302 6 14/268 6 16 (HT); 97 6 4/86 6 4 (HC); 171 6 9/128 6 11 (ST); 193 6 27/287 6 42 (FC).
LINDANE EFFECTS IN DEVELOPING RATS
159 TABLE 1 SUMMARY OF THE EFFECTS OBSERVED IN 15-DAY-OLD RATS 1 h AFTER A SINGLE DOSE OF 20 mg/kg OR 24 h AFTER 7-DAY REPEATED DOSES OF 10 mg/kg LINDANE Lindane Treatment Single
Passive avoidance acquisition Latency time in passive avoidance Spontaneous motor activity Ratio 5-HIAA/serotonin Ratio DOPAC/dopamine
Repeated
Increased Increased
Increased Unchanged
Reduced Increased (HC, FC) Increased (MES)
Increased Decreased (PM, COL, FC) Decreased (MES)/ increased (ST)
See Figs. 1–5 for actual values. See Fig. 4 for abbreviations.
FIG. 5. DOPAC/dopamine ratio in different rat brain regions on postnatal day 15: 1 h after a 20 mg/kg single dose of lindane or 24 h after a 7-day repeated dosing of 10 mg/kg. Values represent the mean 6 SEM of 7–9 animals, normalized by control values (100%). *p , 0.05 (Student’s t-test) compared to control. For abbreviations see Fig. 4. Regional concentrations (ng/g tissue) of metabolite/neurotransmitter in the single dosed control group were: 131 6 17/293 6 16 (MES); 612 6 57/2594 6 126 (ST); 59 6 6/201 6 18 (HT). Concentrations (ng/g tissue) in the repeatedly dosed control animals were: 34 6 2/190 6 12 (MES); 614 6 32/3043 6 180 (ST); 30 6 2/143 6 10 (HT).
ment. The latency time was not altered in the repeated treatment, even though the spontaneous motor activity appeared increased. This suggests that the level of activity did not interfere with the cognitive function. It has been hypothesized that an increased nigrostriatal dopamine metabolism would be in accordance with an increased motor activity (41). In support of the hypothesis would be the increase of DOPAC/dopamine ratio found in the striatum, along with a decrease in the mesencephalon. The system would respond by favoring the release and metabolization of dopamine in the striatum (area of projection), which plays a key role in the control of motor activity. At the same time this would be enhanced by reducing dopamine metabolism in the mesencephalon, where the amine is mainly synthesized, and thus increasing the availability of neurotransmitter in the striatum terminals. On the other hand, serotonin metabolism displayed a general tendency to decrease after the repeated exposure to the neurotoxicant, both in areas of synthesis (pons medulla) and projection (frontal cortex or colliculi). Only the striatum showed a slight nonsignificant increase of the 5-HIAA/serotonin ratio. The differential monoamine status after single and repeated doses may be preferentially linked to the differential motor activity changes. As regards to the anxiogenic effects, rat pups dosed
with 10 mg/kg lindane during the second postnatal week show an increase of ultrasonic vocalizations as do animals dosed once with 20 mg/kg lindane (27). It is tempting to hypothesize that the lindane-induced improvement of the passive avoidance acquisition and the anxiogenic effects, which appear after both single and repeated treatment, are mainly caused by GABAergic disturbances. The immaturity of the rat brain during the second postnatal week, particularly the serotonergic system (14,19), could account for the different response to lindane exposure compared to adults. In addition, the lindane impairment of the myelinization process of the rat throughout the second postnatal week (33) may contribute to the monoamine and behavior profile exhibited by young animals, depending on the age and the treatment schedule. Also in developing rats, a peak of hyperactivity between postnatal days 12–16 has been described after repeated exposure to lindane (28). In adult rats, repeated small doses of lindane have been reported to impair learning capabilities (6) whereas convulsant ones decrease dopamine levels (24). Dieldrin, a related organochloride insecticide, was reported to enhance learning in the adult rat when administrated at very low doses beginning on day 5 of gestation (23). In summary, the present work shows that subconvulsant doses of lindane elicit neurochemical and behavioral effects in developing rats that may be different from those reported in adults, and partially depending on the treatment schedule. It can be concluded that there is some correlation between neurochemical and behavioral data after lindane exposure. Imbalance of both GABA and monoaminergic systems may be the basis of the behavioral disturbances observed in the immature rat. Monoamine alterations may cause the differential acute vs. repeated dosing behavior disturbances, whereas changes of GABAergic function perhaps contribute to the improved passive avoidance retention. Finally, the results support the idea that the limbic and motor systems are major targets for lindane in the central nervous system. ACKNOWLEDGEMENTS
This work was supported by research grants SAF94-0076 and SAF96-0129 from the Spanish Comisión Interministerial de Ciencia y Tecnología (CICYT). The authors gratefully acknowledge the expert technical assistance of C. Cleries and E. Bustamante.
REFERENCES 1. Abalis, I. M.; Eldefrawi, M. E.; Eldefrawi, A. T.: High-affinity binding of cyclodiene insecticides and g-hexachlorocyclohexane
to g-aminobutyric acid receptors of rat brain. Pestic. Biochem. Physiol. 24:95–102; 1985.
160 2. Artigas, F.; Martínez, E.; Camón, L.; Rodríguez-Farré, E.: Synthesis and utilization of neurotransmitters: Actions of subconvulsant doses of hexachlorocyclohexane isomers on brain monoamines. Toxicology 49:49–55; 1988. 3. Brioni, J. D.; McGaugh, J. L.: Posttraining administration of GABAergic antagonists enhances retention of aversively motivated tasks. Psychopharmacology (Berlin) 96:505–510; 1985. 4. Camón, L.; Martínez, E; Artigas, F.; Solà, C.; Rodríguez-Farré E.: The effect of nonconvulsant doses of lindane on temperature and body weight. Toxicology 49:389–394; 1988. 5. Castellano, C.; Introini-Collison, I. B.; McGaugh, J. L.: Interaction of beta-endorphin and GABAergic drugs in the regulation of memory storage. Behav. Neural Biol. 60:123–128; 1993. 6. Dési, I.: Neurotoxicological effects of small quantities of lindane. Int. Arch. Arbeitsmed. 33:153–162; 1974. 7. Dourish, C. T.; Hutson, P. H.; Curzon, G.: Putative anxiolytic 8-OH-DPAT, buspirone and TVX Q 7821 are agonist at 5-HT1A autoreceptors in the raphé nuclei. Trends Pharmacol. Sci. 17:212– 214; 1986. 8. Ghiasuddin, S. M.; Matsumura, F.: Inhibition of g-aminobutyric acid (GABA)-induced chloride uptake by g-BHC and heptachlorepoxide. Comp. Biochem. Physiol. [C] 73:141–144; 1982. 9. Gilbert, M. E.: Repeated exposure to lindane leads to behavioral sensitization and facilitates electrical kindling. Neurotoxicol. Teratol. 17:131–141; 1995. 10. Giménez-Llort, L.; Martínez, E; Ferré, S.: Dopamine-independent and adenosine-dependent mechanisms involved in the effects of N-methyl-D-aspartate on motor activity in mice. Eur. J. Pharmacol. 275:171–177; 1995. 11. Hijzen, T. H.; Slagen, J. L.: Effects of midazolam, DMCM and lindane on potentiated startle in the rat. Psychopharmacology (Berlin) 99:362–365; 1989. 12. Joy, R. M.: Chlorinated hydrocarbon insecticides. In: Ecobichon, D. L.; Joy, R. M., eds. Pesticides and neurological diseases. Boca Raton: CRC Press; 1994:81–170. 13. Joy, R. M.; Stark, L. G.; Albertson, T. E.: Proconvulsant effects of lindane: Enhancement of amygdaloid kindling in the rat. Neurobehav. Toxicol. Teratol. 4:347–354; 1982. 14. Lidov, H. G. W.; Milliver, M E.: An immunohistochemical study of serotonin neurons development in the rat: Ascending pathways and terminal fields. Brain Res. Bull. 8:389–430; 1982. 15. Llorens, J.; Suñol, C.; Tusell, J. M.; Rodríguez-Farré, E.: Evidence for acute tolerance to the behavioral effects of lindane: Concomitant changes in regional monoamine status. Neurotoxicology 12:697–706; 1991. 16. Llorens, J.; Tusell, J. M.; Suñol, C.; Rodríguez-Farré, E.: Effects of lindane on spontaneous behavior of rats analyzed by multivariate statistics. Neurotoxicol. Teratol. 11:145–151; 1989. 17. Llorens, J.; Tusell, J. M.; Suñol, C.; Rodríguez-Farré, E.: On the effects of lindane on the plus maze model of anxiety. Neurotoxicol. Teratol. 12:643–647; 1990. 18. Llorens, J.; Tusell, J. M.; Suñol, C.; Rodríguez-Farré, E.: Repeated lindane exposure in the rat results in changes in spontaneous motor activity at 2 weeks postexposure. Toxicol. Lett. 61:265–274; 1992. 19. Marbry, P. D.; Campbell, B. A.: Ontogeny of serotonergic inhibition of behavioral arousal in the rat. J. Comp. Physiol. Psychol. 86:193–201; 1974. 20. Matsumura, F.; Ghiasuddin, S. M.: Evidence for similarities between cyclodiene type insecticides and picrotoxin in their action mechanisms. J. Environ. Sci. Health B18:1–14; 1983. 21. Moser, P. C.; Tricklebank, M. D.; Middlemiss, D. N.; Mir, A. K.; Hibert, M. F.; Fozar, J. R.: Characterization of MDL 73005EF as a 5-HT1A selective ligand and its effects in animal models of anxiety: Comparison with buspirone, 8-OH-DPAT and diazepam. Br. J. Pharmacol. 99:343–349; 1990. 22. Obata, T.; Yamamura, H. I.; Malatynska, E.; Ikeda, M.; Laird, H.; Palmer, C. J.; Casida, J. E.: Modulation of gamma-aminobutyricstimulated chloride influx by bicycloorthocarboxylates, bycyclophosphorus esters, polychlorocycloalkanes and other cage convulsants. J. Pharmacol. Exp. Ther. 244:802–806; 1988. 23. Olson, K. L.; Bousch, G. M.; Matsumura, F.: Pre- and postnatal
RIVERA ET AL.
24. 25. 26.
27. 28. 29.
30.
31. 32.
33. 34. 35.
36. 37.
38. 39. 40.
41.
42. 43.
exposure to dieldrin: Persistent stimulatory and behavioral effects. Pestic. Biochem. Physiol. 13:20–33; 1980. Ortiz-Martínez, A.; Martínez-Conde, E.: The neurotoxic effects of lindane at acute and subchronic dosages. Ecotoxicol. Environ. Safety 30:101–105; 1995. Pomés, A.; Rodríguez-Farré, E.; Suñol, C.: Inhibition of t-butylbicyclophosphorothionate binding by convulsant agents in primary cultures of cerebellar neurons. Dev. Brain Res. 73:85–90; 1993. Pomés, A.; Rodríguez-Farré, E.; Suñol, C.: Disruption of GABAdependent chloride flux by cyclodienes and hexachlorocyclohexanes in primary cultures of cortical neurons. J. Pharmacol. Exp. Ther. 271:1616–1623; 1994. Rivera, S.; Sanfeliu, C.; García, F.; Comellas, F.; Rodríguez-Farré E.: Increase of rat pup ultrasonic isolation calls induced by lindane. Neurotoxicology 13:235–240; 1992. Rivera, S.; Sanfeliu, C.; Rodríguez-Farré, E.: Behavioral changes induced in developing rats by an early postnatal exposure to lindane. Neurotoxicol. Teratol. 12:591–595; 1990. Rivera, S.; Sanfeliu, C.; Rodríguez-Farré, E.: Changes in regional brain 2-14C-deoxyglucose uptake induced in postnatal developing rats by single and repeated nonconvulsant doses of lindane. Pestic. Biochem. Physiol. 43:241–252; 1992. Rivera, S.; Sanfeliu, C.; Suñol, C.; Rodríguez-Farré, E.: Regional effects on the cerebral concentration of noradrenaline, serotonin and dopamine in suckling rats after a single dose of lindane. Toxicology 69:43–54; 1991. Sanfeliu, C.; Camón, L.; Martínez, E.; Solà, C.; Artigas, F.; Rodríguez-Farré, E.: Regional distribution of lindane in rat brain. Toxicology 49:189–196; 1988. Sanfeliu, C.; Solà, C.; Camón, L.; Martínez, E.; Rodríguez-Farré, E.: Regional changes in brain 2-14C-deoxyglucose uptake induced by convulsant and nonconvulsant doses of lindane. Neurotoxicology 10:727–742; 1989. Serrano, M. T.; Vendrell, M.; Rivera, S.; Serratosa, J.; RodríguezFarré, E.: Effects of lindane on the myelination process in the rat. Neurotoxicol. Teratol. 12:577–583; 1990. Smith, A. G.: Chlorinated hydrocarbon insecticides. In: Hayes, W. J.; Laws, E. R., eds. Handbook of pesticide toxicology. San Diego: Academic Press; 1991:731–915. Solà, C.; Martínez, E.; Camón, L.; Pazos, A.; Rodríguez-Farré, E.: Lindane administration to the rat induces modifications in the regional cerebral binding of [3H]muscimol, [3H]flunitrazepam, and t-[3S]butylbicyclophosphorothionate: An autoradiographic study. J. Neurochem. 60:1821–1834; 1993. Solà, C.; Planas, A.M.; Camón, L.; Martínez, E; Rodríguez-Farré E.: Study of regional cerebral blood flow after lindane administration to the rat. Pestic. Biochem. Physiol. 38:1–8; 1990. Suñol, C.; Tusell, J. M.; Gelpí, E.; Rodríguez-Farré, E.: Regional concentrations of GABA, and noradrenaline in brain at onset of seizures induced by lindane (gamma-hexachlorocyclohexane). Neuropharmacology 27:677–681; 1988. Tilson, H. A.; Shaw, S.; McLamb, R. L.: The effects of lindane, DDT, and chlordecone on avoidance responding and seizure activity. Toxicol. Appl. Pharmacol. 88:57–65; 1987. Treit, D.: Ro 15-1788, CGS 8216, picrotoxin, and pentylenetetrazol: Do they antagonize anxiolytic drug effects through an anxiogenic action? Brain Res. Bull. 19:401–405; 1987. Tusell, J. M.; Suñol, C.; Gelpí, E.; Rodríguez-Farré, E.: Relationship between lindane concentration in blood and brain and convulsant response in rats after oral or intraperitoneal administration. Arch. Toxicol. 60:432–437; 1987. van Praag, H. M.; Asnis, G. M.; Kahn, R. S.; Brown, S. L.; Korn, M.; Friedman, J. M.; Wetzler, S.: Monoamines and abnormal behaviour. A multi-aminergic perspective. Br. J. Psychiatry 157: 723–734; 1990. Woolley, D.; Zimmer, L.; Dodge, D.; Swanson, K.: Effects of lindane-type insecticides in mammals: Unsolved problems. Neurotoxicology 6:165–192; 1985. Woolley, D.; Zimmer, L.; Hasan, Z.; Swanson, K.: Do some insecticides and heavy metals produce long-term potentiation in the limbic system? In: Narahashi, T., ed. Cellular and molecular neurotoxicology. New York: Raven Press; 1984:45–69.