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Effects of chronic dysthyroidism on activity and exploration
Physiology & Behavior 77 (2002) 125 – 133
Effects of chronic dysthyroidism on activity and exploration Josefina Sala-Rocaa,*, Maria Assumpcio´ Martı´-Carbonellb, Adriana Garauc, Sonia Darbrab, Ferran Baladab a
Department of Pedagogy Sistema`tica i Social, Universitat Auto`noma de Barcelona, 08193 Bellaterra, Catalonia, Spain b Department of Psychobiology, Universitat Auto`noma de Barcelona, 08193 Bellaterra, Catalonia, Spain c Department of Basic Psychology, Universitat Auto`noma de Barcelona, 08193 Bellaterra, Catalonia, Spain Received 25 June 2001; received in revised form 15 March 2002; accepted 7 June 2002
Abstract The aim of the present study was to determine the influence of thyroid function on the activity and exploratory behaviour of male Wistar rats. Dysthyroidism was induced by adding drugs to their drinking water from the ninth day of gestation. This method is not as stressful as daily thyroxine injections or thyroidectomy, and therefore did not affect the analysed behavioural patterns. After weaning, the drugs were administered to the young rats until the end of the experiment. Activity and exploration were measured using the Boissier test, a light – darkness test and an open-field test when they were 77 days old. In order to verify that the animals’ motor capacity had not been impaired, a psychomotor battery was used. Chronic hyperthyroidism produced a significant increase in activity, but did not affect exploration. On the other hand, hypothyroidism did not affect activity, but did increase exploration. This increase in exploration was observed in activityindependent behavioural parameters, such as head dipping and glancing. D 2002 Elsevier Science Inc. All rights reserved. Keywords: Hypothyroidism; Hyperthyroidism; Activity; Exploration; Methimazole; Thyroxine; Rat
1. Introduction It is widely known that the presence of thyroid hormones in the development of the nervous system is critical and the deficit of these affects behavioural and intellectual capacities. The most severe form of this disorder, and the first to be related to dysthyroidism, is cretinism. Cretinism is a disorder characterized by severe mental retardation, deafness and strabismus, as well as perceptual, language and motor and other problems. However, initial studies demonstrated that early treatment could reverse or palliate these effects. Therefore, in the seventies, many countries began programs for early hypothyroidism detection in order to start the treatment in the first weeks of life. Nevertheless, even when the treatment starts early, children with congenital hypothyroidism frequently have reduced intellectual capacities, and have motor and learning difficulties [1– 3]. At the same time, the treatments used often present secondary problems: an increase in distraction, hyperactiv-
* Corresponding author. Tel.: +34-93-581-3188; fax: +34-93-581-1419. E-mail address: [email protected] (J. Sala-Roca).
ity, irritability, anxiety, depression and social problems [4,5]. In fact, Alvarez et al. [6] found that hyperthyroid children have poor attention, which improved following euthyroidism. Researchers later discovered that excess of thyroid hormone also causes alterations, namely that hyperthyroidism could lead to attention deficit and hyperactivity disorders. The thyroid hormone resistance syndrome, an alteration produced by mutation of the gene that codifies the b thyroid receptor, induces hyperactivity and attention deficit [7,8]. In fact, 70% of children with this syndrome are diagnosed as having deficits in attention deficit-hyperactivity disorders [9]. However, the consequences of dysthyroidism are not limited to the developmental period. Indeed, dysthyroidism has been implicated in adult disorders such as stress, anxiety and depression, as well as motor and intellectual problems. Over the last three decades, several studies using animal models have studied the behavioural effects of dysthyroidism and the effects of hypothyroidism in particular. Activity and exploration are among the most investigated areas of these studies. Nevertheless, most of these experiments have analysed the effects of neonatal hypothyroidism on
0031-9384/02/$ – see front matter D 2002 Elsevier Science Inc. All rights reserved. PII: S 0 0 3 1 - 9 3 8 4 ( 0 2 ) 0 0 8 1 5 - 6
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adult behaviour, but few of them have explored what happens when the hypothyroid condition continues until adulthood (chronic treatment). Furthermore, there have been no studies that have analysed the effects of chronic hyperthyroidism on these behaviours. The results of the experiments that analysed the effects of chronic hypothyroidism on activity seem contradictory because most of them did not take into consideration the ontogeny of behaviour, started treatment postnatally and used different methods to measure activity without differentiating between activity and exploration. Rastogi and Singhal [10] observed that neonatal thyroidectomy decreases spontaneous locomotor activity measured using an activimeter when the rats were 15 –30 days old, but they did not observe effects at 45 and 60 days. Tamasy et al. [11,12] administered PTU to the drinking water from postnatal day 0 and did not find effects on hole board activity between 42 and 50 days, but, when the activity was measured in the wheel, they observed a decrease in activity. But Sobrian et al. [13] found that 75-day-old animals that received a neonatal injection of 131I showed an increase in open-field activity. In contrast, studies using hyt/hyt mice (animals with a genetic deficiency in the responsiveness of the thyroid gland to TSH) found decreased activity between 12 and 120 days [14,15]. A frequent problem with these studies is the type of test used to measure activity and the interpretation of results. Locomotor activity is indistinctly used as a measure of activity and as a measure of exploration in open-field, hole board and other tests. As locomotor activity takes place in a novel environment, it is related to both activity and exploration. In the last decades, studies of dysthyroidism have acquired a new interest because it has been indicated that these hormones could be the mediators in the toxic effect of alcohol and different environmental contaminants, and other adverse environmental conditions such as an iodine insufficiency. Different studies have indicated that thyroid hormone would be implicated in the foetal alcoholism syndrome [16]. On the other hand, several studies suggest that thyroid hormones could be the mediator of neurotoxic effects of environmental contaminants such dioxins, PCB, etc. (for a review, see Brucker-Davis [17]). The aim of the present study was to clarify the effects of dysthyroidism on the activity and exploratory behaviour of male Wistar rats. Knowledge of these effects would be very useful in the elucidation of the causes of the adverse effects of hypothyroidism treatments, such as hyperactivity, and for a better understanding of the hyperactive syndrome. According to data from previous studies, we believe that the effects of dysthyroidism on activity and exploration are different for hypothyroid versus hyperthyroid states: (1) Previous results showed that chronic hypothyroidism decreases emotionality, a trait that influences exploratory behaviour, but emotionality is not affected by chronic hyperthyroidism [18]. It should therefore be expected that
chronic hypothyroidism increases exploration, whereas chronic hyperthyroidism does not. (2) Neonatal hypothyroidism induces a decrease in activity, whereas neonatal hyperthyroidism provokes an increase in such behaviour [19,20]. These effects will probably still occur in adulthood when treatment is continued to this period, because perinatal dysthyroidism induces organizational effects on the developmental nervous system. We have, in fact, observed a decrease in the anxiety of rats that have recovered from perinatal hypothyroidism [21]. This effect also occurs with chronic hypothyroidism [18], although adult hypothyroidism increases anxiety patterns [22,23], indicating that perinatal treatment effects prevail over adult treatment effects.
2. Materials and methods 2.1. Animals and experimental groups Animals were obtained from progenitors that were randomly assigned to three experimental groups with different treatments. Fifty-one male Wistar rats, bred in our laboratory, were used in this experiment. The experimental groups were hyperthyroid (n = 15), hypothyroid (n = 15) and the control group (n = 21). Throughout the experiment, the animals had free access to food and water. There was a 12-h LD cycle (lights on at 08:00 h) and a controlled environment (22 ± 2 C and 40 –60% humidity). 2.2. Procedure Treatments were administered via drinking water to pregnant females from the ninth day of gestation until weaning and were subsequently administered to the young rats until the end of the experiment. Dysthyroidism was induced by adding 20 mg methimazole (Sigma)/100 ml or by adding 0.3 mg L-thyroxine (Sigma)/100 ml to the drinking water. Methimazole is an antithyroidal compound of the tiourelines that block the organic binding of iodide and the coupling of iodothyronines to form T4 and T3 without interfering with peripherical tissue deiodinase activity and that does not have the bitter taste of PTU. We used this method because it is less stressful than daily injections or thyroidectomy, an important factor in behavioural studies. This method has been proved to be effective in the induction of dysthyroidism [18,21]. Behavioural tests started when the rats were 77 days old and finished when they were 82 days old. The tests were administered between 11:00 and 13:00 h. The animals were weighed every week after weaning until they were killed by decapitation on Day 89, so we could collect blood samples in order to analyse T4 levels. The experimental protocol complies with the European Community Council Directive (EEC directive 86/609) for the care and use of laboratory animals, and therefore has the
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approval of the Ethical Committee on Animal and Human Experimentation of the Universitat Auto`noma de Barcelona. 2.3. Behavioural tests 2.3.1. Psychomotor battery To verify that the effect of the treatment on activity and exploration was not due to impaired motor capacity, we evaluated motor capacity and certain elemental reflexes using a psychomotor battery. This battery included: Negative geotaxis: the animal was placed, head downwards, on a net-surface positioned at an angle of 20. Inclined screen: the animal was placed, head upwards, on a net-surface positioned at an angle of 80. Rod-walking: the animal was placed longitudinally over a 3.5 diameter wood cylinder positioned 40 cm above the floor. Wire-hanging: a metal bar was placed against the ventral surface of both forepaws while the animal was held in a vertical position. Measures included in this battery were a test of the latency of rotation on the negative geotaxis and the timing of permanence on the inclined screen, rodwalking and wire-hanging tests. 2.3.2. The light – darkness test This test, which measures exploration [21], was performed in an adapted shuttle box (Lafayette Ins., Mol. 82401-SS) with two compartments, one of which was lit (40 W) while the other was dark. They were separated by a hard wall. There was a 5-cm space under the wall that allowed the animal to move from one compartment to the other. The rat was first placed in the dark compartment, where the latency of compartment change, the number of changes, the number of glances at the other compartment and the accumulated time on the illuminated side were measured for 10 min. 2.3.3. The Boissier test This test, adapted from the Boissier and Simon test for mice [24], evaluates both activity and exploratory behaviour. The rats were placed in the middle of the apparatus and ambulation (the number of floor units crossed), dippings of the head (the number of times the rat introduced its head in the holes as far as the eye line), rearings (the frequency with which the rats stood on their hind legs) and defecations (the number of faecal boluses) were measured for 5 min.
because it is considered that the measurement of ambulation on the first day reflects novelty, whereas measurement of second day ambulation is less affected by novelty. Therefore, we took measurements from the second day to assess activity. The test was performed at the same time every day. 2.4. Hormonal analyses T4 serum levels were measured in order to establish the degree of dysthyroidism induced by our treatment. Animals were killed by decapitation when they were 89 days old, between 10:00 and 11:00 a.m. Blood samples were collected and centrifuged, and serum was immediately frozen and stored at 40 C. T4 serum was determined by radioimmunoassay (reference values 4.5– 12.5 mg/dl). The kit was equipped with standard T4 values ranging from 1 to 24 mg/dl. The antiserum was highly specific for T4. The procedure can detect as little as 0.25 mg/dl. Each sample required a duplicate sample to check reliability. The percentage recovery of the evaluations fell within the standard and acceptable limits for these measures. The coefficient of variation between duplicate samples was always < 5%. 2.5. Statistical analysis The data were analysed using a commercial statistical package (SPSS/PC+). Analysis of variance (ANOVA) was
Table 1 Tests and variables measured Test
Motor competence
Psychomotor battery
Inclined screen time Wire-hanging time Rod-walking time Negative geotaxis latency
Activity
Light – darkness test
Boissier test
2.3.4. The open-field test We used an open-field test equipped with a timer, a pink noise generator (60 dB) and a light (100 W) 1 m above the floor. Ambulation, rearing and defecation were measured for 4 min. This test was performed on 2 consecutive days
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Open-field test
Ambulation Rearing Ambulation second open field Rearing second open field
Exploration
Latency of compartment change Number of changes Number of glances at the other compartment Time in light side Head dipping
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univariate analysis. Therefore, one multivariate analysis of ambulation and rearing, observed in the Boissier and the second open-field tests, was carried out to analyse the effects of chronic dysthyroidism on activity. Another multivariate analysis of the parameters measured in the light– darkness test, ambulation in the first open field and head dipping in the Boissier test, measured the effects on exploration. Although head dipping is a parameter from the Boissier test, it was included in the second analysis because File and Wardill [25] showed that the number of holes explored is a different exploration parameter to locomotor activity (Table 1).
3. Results 3.1. Plasma T4 levels Fig. 1. Box and Jenkins diagram of the T4 plasma levels with quartiles (black line indicates median value).
performed to detect differences in plasma T4 levels and orthogonal contrasts were applied to determine the differences between groups. Differences in body weight were analysed using ANOVA in repeated measures and polynomial contrasts were used to measure the differences in the weight gains associated with development. The effects of dysthyroidism on behaviour were assessed using multivariate analysis of variance (MANOVA) and orthogonal contrast was used to measure the differences between groups. Multivariate analysis was used because it considers covariation between parameters that measure the same attribute and this analysis also gives more reliable results than
There were differences in the plasma T4 levels of the experimental groups [ F(2,44) = 38.28, P < .0001]. The plasma T4 levels of the hypothyroid group were significantly reduced ( P < .0001), while those of the hyperthyroid group were significantly increased ( P < .0001) (Fig. 1). Weight gain was affected by dysthyroidism [ F(20,380) = 106.5, P < .001]. Polynomial contrasts revealed that the slope of the weight increase in the hypothyroid group was lower than that of the control group ( P < .001), while that of the hyperthyroid group was slightly higher ( P < .05) (Fig. 2). 3.2. Motor capacity Multivariate analysis of the variance of the psychomotor battery indicated that the motor capacity of the treated
Fig. 2. Weight evolution from weaning until the end of the experiment. Measures were taken weekly.
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Table 2 Means, S.E.M. and standardized coefficients of discriminant functions generated by MANOVA of behavioural pattern analyzed Hypothyroid group
Control group
Hyperthyroid group
Coefficient discriminant function
Mean ± S.E.M.
Mean ± S.E.M.
Mean ± S.E.M.
Motor capacity and reflexes Time permanency inclined screen Time permanency wire-hanging Time permanency rod-walking Latency negative geotaxis
45.23 ± 4.54 10.99 ± 1.72 1.02 ± 0.29 11.83 ± 2.56
21.59 ± 4.69 8.45 ± 2.02 1.09 ± 0.26 28.94 ± 4.79
5.38 ± 1.47 8.70 ± 1.67 1.55 ± 0.37 33.46 ± 5.10
0.854 0.148 0.085 0.486
Activity Rearings of Boissier test Ambulations of open-field test Ambulations of Boissier test Rearings of open-field test
4.87 ± 1.43 24.67 ± 6.30 10.87 ± 1.57 6.93 ± 1.98
3.29 ± 0.99 24.67 ± 3.46 6.00 ± 1.15 6.86 ± 1.64
9.67 ± 2.25 38.13 ± 5.21 11.07 ± 1.45 9.20 ± 1.52
0.785 0.529 0.320 0.392
22.87 ± 2.50 21.33 ± 2.30 85.47 ± 32.10 346.40 ± 63.26 4.27 ± 1.07
10.23 ± 2.07 9.24 ± 0.90 102.43 ± 21.64 334.05 ± 49.04 5.90 ± 1.28
11.93 ± 2.67 10.73 ± 1.00 52.73 ± 16.78 402.73 ± 67.23 3.60 ± 1.19
0.684 0.687 0.425 0.022 0.741
Exploration Number of head dippings Number of glances Time in illuminated side Latency of change compartment Number of changes Time was measured in seconds.
animals was not impaired. What is more, the hypothyroid animals performed better in this battery than the control [Wilks = 0.617, F(4,45) = 6.99, P < .001] and hyperthyroid [Wilks = 0.479, F(4,45) = 12.23, P < .001] animals. Table 2 shows the group means, S.E.M. and the standardized coefficients of the generated discriminant function. The coefficients indicate that hypothyroid animals remained on the inclined screen longer and rotated more quickly on the negative geotaxis test than the control and hyperthyroid animals.
3.3. Activity The hyperthyroid group was more active than the control group [Wilks = 0.760, F(4,45) = 3.54, P < .05]. This increase was observed in all measured activity parameters. On the other hand, although multivariate analysis did not find significant differences between the hypothyroid and control groups, the hypothyroid animals ambulated more in the Boissier test than the control animals (Table 2). There were no differences in the ambulation of the experimental
Fig. 3. Evolution of ambulations from the first and second open-field test. The figure shows that the evolution of the number of ambulations in hyper- and hypothyroidthyroxine and methimazole groups is the opposite. This different evolution is significant.
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groups on the open-field on the first day, but the hypothyroid animals ambulated more on the first day than on the second, while the hyperthyroid animals ambulated more on the second day than on the first. These differences (first day scores minus second day scores) reached statistical significance ( P < .03) (Fig. 3). 3.4. Exploration The multivariate analysis showed that the hypothyroid group explored more than the control [Wilks = 0.551, F(5,44) = 7.17, P < .001] and hyperthyroid groups [Wilks = 0.676, F(5,44) = 4.22, P < .01]. The standardized coefficients indicate that the generated discriminant function is polarized. Therefore, the variables used are related to two kinds of exploration: static exploration (dippings of the head in the Boissier test, visual exploration of the other compartment and the accumulated time on the illuminated side of the light – darkness test) and active exploration (changes of compartment and latency in the light– darkness test) (Table 2).
4. Discussion Plasma thyroxin levels and the body weight curve confirmed to us that the treatments used were effective. The effects of hypothyroidism on body weight were dramatic. Nevertheless, the animals showed no evidence of physical alteration. Veterinary controls did not reveal any health problems. In fact, the psychomotor test did not show that the motor capacity of the treated animals was impaired. These are amazing results because literature indicates that neonatal or chronic hypothyroidism provokes severe neurological defects. Nevertheless, previous studies [18,21] have not indicated any serious defects in animals treated perinatally with methimazole in their drinking water. These results suggest that this treatment, despite being chronic, could be considered mild. Chronic hypothyroidism does not seem to affect activity. The increased ambulation of the hypothyroid group in the Boissier test was probably more related to the exploration of the new environment rather than to activity. In fact, no increase in ambulation was observed on the second day of the open-field test. As stated earlier in Section 2, measures from the second day of this test are less contaminated by exploration. Our results indicate that the hypothyroid animals in the open-field test ambulated more on the first day, i.e. when it was a novel environment, whereas the hyperthyroid animals ambulated more on the second day, i.e. when it was a known environment. These results suggest that the characteristic hyperactivity of rehabilitated animals from congenital or perinatal hypothyroidism [11,12,19,21,26 – 28,31] does not appear if the hypothyroid state continues. In previous studies, we have observed that adult hypothyroidism does not affect activity
either [22,23]. Our present data differs from the results of another study [13] in which animals were radiothyroidectomized 4 h after delivery. In this study, it was found that chronic hypothyroidism increased locomotor activity, but there was no normal increase in deaths or illness in severe treatments reported, so it is not unlikely that certain compensatory processes have taken place. It has also been reported that hypothyroidism affects the activity circadian rhythm, but the administration of thyroxine to untreated animals does not [29]. Therefore, it seems that the control of the circadian effects could be relevant in hypothyroid animals. This suggests that the absence of any effects of hypothyroidism on activity in our experiment could be influenced by the moment at which the animals were tested. In fact, Schull et al. [30] showed that the activity levels of thyro-parathyroidectomized animals increase more abruptly as it gets dark than sham-operated did. Nevertheless, as mentioned earlier, the administration of thyroxine does not affect the activity circadian rhythm, so it seems that the increase in the activity of the hyperthyroid group observed in our experiment is not related to the activity circadian rhythm. Different biological substrates could be implicated in thyroid hormones effect on activity. In fact, hypothyroidism depresses dopaminergic [10,32 – 35] and noradrenergic [10,36,37] activity. There is considerable evidence to indicate that novelty-seeking behaviour involves the activation of the mesolimbic DA system. It appears that D1 receptors may mediate the incentive rewarding properties of novelty, whereas D2 receptors may be more important in mediating locomotor performance per se [38]. It will be interesting to delimit whether the effects observed for hypo- and hyperthyroidism are the result of the thyroid action over D1 or D2 receptors. The perinatal period seems extremely vulnerable to hypothyroidism with respect to changes in locomotor activity. In addition, chronic but not acute dysthyroidism modifies utilization of dopamine [39,40]. Hypothyroidism induces the loss of dopamine receptors in the developing brain [34], which might sensitise them. If this sensitisation is permanent, continued hypothyroidism could counteract the effects of chronic dysthyroidism on the dopaminergic system. This hypothesis would account for the lack of effects on activity observed in chronic hypothyroid animals. However, if normal thyroid levels are recovered, and therefore the activity of the noradrenergic and dopaminergic systems too, the permanent sensitisation of the dopamine receptors would lead to an increase in the functionality of these systems. This could explain the increase in activity in animals recovered from perinatal hypothyroidism [11,12, 19,21,26 – 28,31], some effects of the therapies [4,5] and the hyperactivity in the thyroid hormone resistance syndrome [7,8]. At the beginning of the behavioural assessment, motor capacity and basic reflexes were evaluated so that impaired motor capacity could be rejected as a causal factor of the results obtained in the activity and exploration tests. The
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results in the psychomotor battery confirmed that the basic reflexes analysed were correctly developed and the motor capacity of the animals was not impaired. In fact, hypothyroidism delays and hyperthyroidism accelerates the appearance of these reflexes, but both groups of animals eventually develop them normally [41]. Interestingly, the hypothyroid group performed better in this battery than both the control and the hyperthyroid groups. Namely, they remained longer on the inclined screen test and turned more quickly in the negative geotaxis test. This better performance could well be related to behavioural differences in coping with situations that are potentially anxiogenic because of the danger of falling or novelty. In fact, we found that perinatal and chronic hypothyroidism induced an anxiolytic behavioural pattern measured by the plus maze in adulthood [18,21,22]. Our results showed that chronic hyperthyroidism increases activity. As far as we know, there is no data on the effects of chronic hyperthyroidism on activity from the perinatal period to adulthood. Neonatal hyperthyroidism seems to increase activity [42], but this hyperactivity does not continue into adulthood when animals recover their normal state [21,43]. However, it has been pointed out that single intraperitoneal injections of high dosages of thyroid hormones in the neonatal period cause hyperactivity in adulthood [44 –46]. Adult hyperthyroidism also seems to produce an increase in activity [22,36,42], although there are discrepant results [10]. The increase in dopamine [42,47] or noradrenaline [42,48] activity is also believed to be the mechanism implicated in the hyperactivity caused by hyperthyroidism. However, in this case, the effect could be more activational (i.e. dependent on current hormone levels) than the organizational effects observed in hypothyroidism. Nevertheless, as mentioned earlier, high dosages of T4 or T3 on the third or fourth day after delivery provokes permanent hyperactive behaviour [44 –46]. It is therefore likely that there are short periods of time, in which the organism is highly sensitive to variations in levels of thyroid hormone, and these could affect the organization of the pituitary – thyroid axis. Nevertheless, this effect may well be more closely related to the high dosages of thyroid hormones than to the affected period. Related to exploration behaviour, the results obtained with the light– darkness test suggest that the discriminant function measured static exploration by considering the fact that variables such as glances, the number of head dippings and the time spent on the illuminated side had a positive influence on the function, while the number of compartmental changes influenced it negatively. The hypothyroid animals explored more than the control and hyperthyroid animals, although they were not as active as the hyperthyroid animals. In fact, as stated above, hypothyroid animals ambulated more on the open field during the first day than on the second day. The decrease in ambulation on the second day could be related to familiarity with the
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environment. A similar effect in the light– darkness test was observed in adult animals that had recovered from perinatal hypothyroidism [21], but, in this test, adult hypothyroid animals did not explore more than the control animals did [22]. This data suggests that the effect on exploration would be an organizational effect, limited to the perinatal period. This could be related to the anxiolytic behavioural pattern observed in perinatal hypothyroidism [21]. Brucker-Davis [17] after an exhaustive revision indicated that several categories of synthetic chemicals are suspected to be thyroid disruptors or modulators, potentially altering the thyroid economy at multiple levels. In fact, data available from two accidental polychlorinated biphenyl (PCB) poisonings in Japan and in Taiwan indicate that neurodevelopmental deficit, behaviour and cutaneous signs were the dominant clinical features. It is interesting that Yucheng patients (from a Taiwan accident), who presented with an increase of free thyroxine and T3, were described as hyperactive, which is coincident with the results of our study, and with the hypothesis that the attention deficithyperactivity disorder could be related to hyperthyroidism. All human studies at different levels of exposure and contamination show some degree of neurodevelopment impairment. However, the impact on humans may depend on timing, dose and duration of exposure, synergy between chemicals, as well as genetic and immune status predisposing the exposed person to thyroid diseases [17]. In conclusion, the results of this experiment indicate that chronic thyroid deficiency increases exploration but not activity. In contrast, chronic hyperthyroidism increases activity but not exploration. These results agree with the hypothesized effects of chronic dysthyroidism on exploratory behaviour. The hypothesized effects on activity behaviour were only confirmed in the hyperthyroid group, whereas no differences in this behaviour were observed in the hypothyroid group. This suggests that the effects of perinatal hypothyroidism in activity could be counteracted by the effects of adult hypothyroidism. Acknowledgements We gratefully acknowledge Eva Estebanez’s collaboration and Dr. Dolors Ribas’ advice in our statistical analysis. References [1] Rochiccioli P, Roge´ B, Alexandre F, Tauber MT. School achievement in children with hypothyroidism detected at birth and search for predictive factors. Horm Res 1992;38:236 – 40. [2] Gottschalk B, Richman RA, Lewandowski L. Subtle speech and motor deficits of children with congenital hypothyroid treated early. Dev Med Child Neurol 1994;36:216 – 20. [3] Derksen-Lubsen G, Verkerk PH. Neuropsychologic development in early treated congenital hyperthyroidism: analysis of literature data. Pediatr Res 1996;39(3):561 – 6.
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Effects of chronic dysthyroidism on activity and exploration Josefina Sala-Rocaa,*, Maria Assumpcio´ Martı´-Carbonellb, Adriana Garauc, Sonia Darbrab, Ferran Baladab a
Department of Pedagogy Sistema`tica i Social, Universitat Auto`noma de Barcelona, 08193 Bellaterra, Catalonia, Spain b Department of Psychobiology, Universitat Auto`noma de Barcelona, 08193 Bellaterra, Catalonia, Spain c Department of Basic Psychology, Universitat Auto`noma de Barcelona, 08193 Bellaterra, Catalonia, Spain Received 25 June 2001; received in revised form 15 March 2002; accepted 7 June 2002
Abstract The aim of the present study was to determine the influence of thyroid function on the activity and exploratory behaviour of male Wistar rats. Dysthyroidism was induced by adding drugs to their drinking water from the ninth day of gestation. This method is not as stressful as daily thyroxine injections or thyroidectomy, and therefore did not affect the analysed behavioural patterns. After weaning, the drugs were administered to the young rats until the end of the experiment. Activity and exploration were measured using the Boissier test, a light – darkness test and an open-field test when they were 77 days old. In order to verify that the animals’ motor capacity had not been impaired, a psychomotor battery was used. Chronic hyperthyroidism produced a significant increase in activity, but did not affect exploration. On the other hand, hypothyroidism did not affect activity, but did increase exploration. This increase in exploration was observed in activityindependent behavioural parameters, such as head dipping and glancing. D 2002 Elsevier Science Inc. All rights reserved. Keywords: Hypothyroidism; Hyperthyroidism; Activity; Exploration; Methimazole; Thyroxine; Rat
1. Introduction It is widely known that the presence of thyroid hormones in the development of the nervous system is critical and the deficit of these affects behavioural and intellectual capacities. The most severe form of this disorder, and the first to be related to dysthyroidism, is cretinism. Cretinism is a disorder characterized by severe mental retardation, deafness and strabismus, as well as perceptual, language and motor and other problems. However, initial studies demonstrated that early treatment could reverse or palliate these effects. Therefore, in the seventies, many countries began programs for early hypothyroidism detection in order to start the treatment in the first weeks of life. Nevertheless, even when the treatment starts early, children with congenital hypothyroidism frequently have reduced intellectual capacities, and have motor and learning difficulties [1– 3]. At the same time, the treatments used often present secondary problems: an increase in distraction, hyperactiv-
* Corresponding author. Tel.: +34-93-581-3188; fax: +34-93-581-1419. E-mail address: [email protected] (J. Sala-Roca).
ity, irritability, anxiety, depression and social problems [4,5]. In fact, Alvarez et al. [6] found that hyperthyroid children have poor attention, which improved following euthyroidism. Researchers later discovered that excess of thyroid hormone also causes alterations, namely that hyperthyroidism could lead to attention deficit and hyperactivity disorders. The thyroid hormone resistance syndrome, an alteration produced by mutation of the gene that codifies the b thyroid receptor, induces hyperactivity and attention deficit [7,8]. In fact, 70% of children with this syndrome are diagnosed as having deficits in attention deficit-hyperactivity disorders [9]. However, the consequences of dysthyroidism are not limited to the developmental period. Indeed, dysthyroidism has been implicated in adult disorders such as stress, anxiety and depression, as well as motor and intellectual problems. Over the last three decades, several studies using animal models have studied the behavioural effects of dysthyroidism and the effects of hypothyroidism in particular. Activity and exploration are among the most investigated areas of these studies. Nevertheless, most of these experiments have analysed the effects of neonatal hypothyroidism on
0031-9384/02/$ – see front matter D 2002 Elsevier Science Inc. All rights reserved. PII: S 0 0 3 1 - 9 3 8 4 ( 0 2 ) 0 0 8 1 5 - 6
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adult behaviour, but few of them have explored what happens when the hypothyroid condition continues until adulthood (chronic treatment). Furthermore, there have been no studies that have analysed the effects of chronic hyperthyroidism on these behaviours. The results of the experiments that analysed the effects of chronic hypothyroidism on activity seem contradictory because most of them did not take into consideration the ontogeny of behaviour, started treatment postnatally and used different methods to measure activity without differentiating between activity and exploration. Rastogi and Singhal [10] observed that neonatal thyroidectomy decreases spontaneous locomotor activity measured using an activimeter when the rats were 15 –30 days old, but they did not observe effects at 45 and 60 days. Tamasy et al. [11,12] administered PTU to the drinking water from postnatal day 0 and did not find effects on hole board activity between 42 and 50 days, but, when the activity was measured in the wheel, they observed a decrease in activity. But Sobrian et al. [13] found that 75-day-old animals that received a neonatal injection of 131I showed an increase in open-field activity. In contrast, studies using hyt/hyt mice (animals with a genetic deficiency in the responsiveness of the thyroid gland to TSH) found decreased activity between 12 and 120 days [14,15]. A frequent problem with these studies is the type of test used to measure activity and the interpretation of results. Locomotor activity is indistinctly used as a measure of activity and as a measure of exploration in open-field, hole board and other tests. As locomotor activity takes place in a novel environment, it is related to both activity and exploration. In the last decades, studies of dysthyroidism have acquired a new interest because it has been indicated that these hormones could be the mediators in the toxic effect of alcohol and different environmental contaminants, and other adverse environmental conditions such as an iodine insufficiency. Different studies have indicated that thyroid hormone would be implicated in the foetal alcoholism syndrome [16]. On the other hand, several studies suggest that thyroid hormones could be the mediator of neurotoxic effects of environmental contaminants such dioxins, PCB, etc. (for a review, see Brucker-Davis [17]). The aim of the present study was to clarify the effects of dysthyroidism on the activity and exploratory behaviour of male Wistar rats. Knowledge of these effects would be very useful in the elucidation of the causes of the adverse effects of hypothyroidism treatments, such as hyperactivity, and for a better understanding of the hyperactive syndrome. According to data from previous studies, we believe that the effects of dysthyroidism on activity and exploration are different for hypothyroid versus hyperthyroid states: (1) Previous results showed that chronic hypothyroidism decreases emotionality, a trait that influences exploratory behaviour, but emotionality is not affected by chronic hyperthyroidism [18]. It should therefore be expected that
chronic hypothyroidism increases exploration, whereas chronic hyperthyroidism does not. (2) Neonatal hypothyroidism induces a decrease in activity, whereas neonatal hyperthyroidism provokes an increase in such behaviour [19,20]. These effects will probably still occur in adulthood when treatment is continued to this period, because perinatal dysthyroidism induces organizational effects on the developmental nervous system. We have, in fact, observed a decrease in the anxiety of rats that have recovered from perinatal hypothyroidism [21]. This effect also occurs with chronic hypothyroidism [18], although adult hypothyroidism increases anxiety patterns [22,23], indicating that perinatal treatment effects prevail over adult treatment effects.
2. Materials and methods 2.1. Animals and experimental groups Animals were obtained from progenitors that were randomly assigned to three experimental groups with different treatments. Fifty-one male Wistar rats, bred in our laboratory, were used in this experiment. The experimental groups were hyperthyroid (n = 15), hypothyroid (n = 15) and the control group (n = 21). Throughout the experiment, the animals had free access to food and water. There was a 12-h LD cycle (lights on at 08:00 h) and a controlled environment (22 ± 2 C and 40 –60% humidity). 2.2. Procedure Treatments were administered via drinking water to pregnant females from the ninth day of gestation until weaning and were subsequently administered to the young rats until the end of the experiment. Dysthyroidism was induced by adding 20 mg methimazole (Sigma)/100 ml or by adding 0.3 mg L-thyroxine (Sigma)/100 ml to the drinking water. Methimazole is an antithyroidal compound of the tiourelines that block the organic binding of iodide and the coupling of iodothyronines to form T4 and T3 without interfering with peripherical tissue deiodinase activity and that does not have the bitter taste of PTU. We used this method because it is less stressful than daily injections or thyroidectomy, an important factor in behavioural studies. This method has been proved to be effective in the induction of dysthyroidism [18,21]. Behavioural tests started when the rats were 77 days old and finished when they were 82 days old. The tests were administered between 11:00 and 13:00 h. The animals were weighed every week after weaning until they were killed by decapitation on Day 89, so we could collect blood samples in order to analyse T4 levels. The experimental protocol complies with the European Community Council Directive (EEC directive 86/609) for the care and use of laboratory animals, and therefore has the
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approval of the Ethical Committee on Animal and Human Experimentation of the Universitat Auto`noma de Barcelona. 2.3. Behavioural tests 2.3.1. Psychomotor battery To verify that the effect of the treatment on activity and exploration was not due to impaired motor capacity, we evaluated motor capacity and certain elemental reflexes using a psychomotor battery. This battery included: Negative geotaxis: the animal was placed, head downwards, on a net-surface positioned at an angle of 20. Inclined screen: the animal was placed, head upwards, on a net-surface positioned at an angle of 80. Rod-walking: the animal was placed longitudinally over a 3.5 diameter wood cylinder positioned 40 cm above the floor. Wire-hanging: a metal bar was placed against the ventral surface of both forepaws while the animal was held in a vertical position. Measures included in this battery were a test of the latency of rotation on the negative geotaxis and the timing of permanence on the inclined screen, rodwalking and wire-hanging tests. 2.3.2. The light – darkness test This test, which measures exploration [21], was performed in an adapted shuttle box (Lafayette Ins., Mol. 82401-SS) with two compartments, one of which was lit (40 W) while the other was dark. They were separated by a hard wall. There was a 5-cm space under the wall that allowed the animal to move from one compartment to the other. The rat was first placed in the dark compartment, where the latency of compartment change, the number of changes, the number of glances at the other compartment and the accumulated time on the illuminated side were measured for 10 min. 2.3.3. The Boissier test This test, adapted from the Boissier and Simon test for mice [24], evaluates both activity and exploratory behaviour. The rats were placed in the middle of the apparatus and ambulation (the number of floor units crossed), dippings of the head (the number of times the rat introduced its head in the holes as far as the eye line), rearings (the frequency with which the rats stood on their hind legs) and defecations (the number of faecal boluses) were measured for 5 min.
because it is considered that the measurement of ambulation on the first day reflects novelty, whereas measurement of second day ambulation is less affected by novelty. Therefore, we took measurements from the second day to assess activity. The test was performed at the same time every day. 2.4. Hormonal analyses T4 serum levels were measured in order to establish the degree of dysthyroidism induced by our treatment. Animals were killed by decapitation when they were 89 days old, between 10:00 and 11:00 a.m. Blood samples were collected and centrifuged, and serum was immediately frozen and stored at 40 C. T4 serum was determined by radioimmunoassay (reference values 4.5– 12.5 mg/dl). The kit was equipped with standard T4 values ranging from 1 to 24 mg/dl. The antiserum was highly specific for T4. The procedure can detect as little as 0.25 mg/dl. Each sample required a duplicate sample to check reliability. The percentage recovery of the evaluations fell within the standard and acceptable limits for these measures. The coefficient of variation between duplicate samples was always < 5%. 2.5. Statistical analysis The data were analysed using a commercial statistical package (SPSS/PC+). Analysis of variance (ANOVA) was
Table 1 Tests and variables measured Test
Motor competence
Psychomotor battery
Inclined screen time Wire-hanging time Rod-walking time Negative geotaxis latency
Activity
Light – darkness test
Boissier test
2.3.4. The open-field test We used an open-field test equipped with a timer, a pink noise generator (60 dB) and a light (100 W) 1 m above the floor. Ambulation, rearing and defecation were measured for 4 min. This test was performed on 2 consecutive days
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Open-field test
Ambulation Rearing Ambulation second open field Rearing second open field
Exploration
Latency of compartment change Number of changes Number of glances at the other compartment Time in light side Head dipping
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univariate analysis. Therefore, one multivariate analysis of ambulation and rearing, observed in the Boissier and the second open-field tests, was carried out to analyse the effects of chronic dysthyroidism on activity. Another multivariate analysis of the parameters measured in the light– darkness test, ambulation in the first open field and head dipping in the Boissier test, measured the effects on exploration. Although head dipping is a parameter from the Boissier test, it was included in the second analysis because File and Wardill [25] showed that the number of holes explored is a different exploration parameter to locomotor activity (Table 1).
3. Results 3.1. Plasma T4 levels Fig. 1. Box and Jenkins diagram of the T4 plasma levels with quartiles (black line indicates median value).
performed to detect differences in plasma T4 levels and orthogonal contrasts were applied to determine the differences between groups. Differences in body weight were analysed using ANOVA in repeated measures and polynomial contrasts were used to measure the differences in the weight gains associated with development. The effects of dysthyroidism on behaviour were assessed using multivariate analysis of variance (MANOVA) and orthogonal contrast was used to measure the differences between groups. Multivariate analysis was used because it considers covariation between parameters that measure the same attribute and this analysis also gives more reliable results than
There were differences in the plasma T4 levels of the experimental groups [ F(2,44) = 38.28, P < .0001]. The plasma T4 levels of the hypothyroid group were significantly reduced ( P < .0001), while those of the hyperthyroid group were significantly increased ( P < .0001) (Fig. 1). Weight gain was affected by dysthyroidism [ F(20,380) = 106.5, P < .001]. Polynomial contrasts revealed that the slope of the weight increase in the hypothyroid group was lower than that of the control group ( P < .001), while that of the hyperthyroid group was slightly higher ( P < .05) (Fig. 2). 3.2. Motor capacity Multivariate analysis of the variance of the psychomotor battery indicated that the motor capacity of the treated
Fig. 2. Weight evolution from weaning until the end of the experiment. Measures were taken weekly.
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Table 2 Means, S.E.M. and standardized coefficients of discriminant functions generated by MANOVA of behavioural pattern analyzed Hypothyroid group
Control group
Hyperthyroid group
Coefficient discriminant function
Mean ± S.E.M.
Mean ± S.E.M.
Mean ± S.E.M.
Motor capacity and reflexes Time permanency inclined screen Time permanency wire-hanging Time permanency rod-walking Latency negative geotaxis
45.23 ± 4.54 10.99 ± 1.72 1.02 ± 0.29 11.83 ± 2.56
21.59 ± 4.69 8.45 ± 2.02 1.09 ± 0.26 28.94 ± 4.79
5.38 ± 1.47 8.70 ± 1.67 1.55 ± 0.37 33.46 ± 5.10
0.854 0.148 0.085 0.486
Activity Rearings of Boissier test Ambulations of open-field test Ambulations of Boissier test Rearings of open-field test
4.87 ± 1.43 24.67 ± 6.30 10.87 ± 1.57 6.93 ± 1.98
3.29 ± 0.99 24.67 ± 3.46 6.00 ± 1.15 6.86 ± 1.64
9.67 ± 2.25 38.13 ± 5.21 11.07 ± 1.45 9.20 ± 1.52
0.785 0.529 0.320 0.392
22.87 ± 2.50 21.33 ± 2.30 85.47 ± 32.10 346.40 ± 63.26 4.27 ± 1.07
10.23 ± 2.07 9.24 ± 0.90 102.43 ± 21.64 334.05 ± 49.04 5.90 ± 1.28
11.93 ± 2.67 10.73 ± 1.00 52.73 ± 16.78 402.73 ± 67.23 3.60 ± 1.19
0.684 0.687 0.425 0.022 0.741
Exploration Number of head dippings Number of glances Time in illuminated side Latency of change compartment Number of changes Time was measured in seconds.
animals was not impaired. What is more, the hypothyroid animals performed better in this battery than the control [Wilks = 0.617, F(4,45) = 6.99, P < .001] and hyperthyroid [Wilks = 0.479, F(4,45) = 12.23, P < .001] animals. Table 2 shows the group means, S.E.M. and the standardized coefficients of the generated discriminant function. The coefficients indicate that hypothyroid animals remained on the inclined screen longer and rotated more quickly on the negative geotaxis test than the control and hyperthyroid animals.
3.3. Activity The hyperthyroid group was more active than the control group [Wilks = 0.760, F(4,45) = 3.54, P < .05]. This increase was observed in all measured activity parameters. On the other hand, although multivariate analysis did not find significant differences between the hypothyroid and control groups, the hypothyroid animals ambulated more in the Boissier test than the control animals (Table 2). There were no differences in the ambulation of the experimental
Fig. 3. Evolution of ambulations from the first and second open-field test. The figure shows that the evolution of the number of ambulations in hyper- and hypothyroidthyroxine and methimazole groups is the opposite. This different evolution is significant.
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groups on the open-field on the first day, but the hypothyroid animals ambulated more on the first day than on the second, while the hyperthyroid animals ambulated more on the second day than on the first. These differences (first day scores minus second day scores) reached statistical significance ( P < .03) (Fig. 3). 3.4. Exploration The multivariate analysis showed that the hypothyroid group explored more than the control [Wilks = 0.551, F(5,44) = 7.17, P < .001] and hyperthyroid groups [Wilks = 0.676, F(5,44) = 4.22, P < .01]. The standardized coefficients indicate that the generated discriminant function is polarized. Therefore, the variables used are related to two kinds of exploration: static exploration (dippings of the head in the Boissier test, visual exploration of the other compartment and the accumulated time on the illuminated side of the light – darkness test) and active exploration (changes of compartment and latency in the light– darkness test) (Table 2).
4. Discussion Plasma thyroxin levels and the body weight curve confirmed to us that the treatments used were effective. The effects of hypothyroidism on body weight were dramatic. Nevertheless, the animals showed no evidence of physical alteration. Veterinary controls did not reveal any health problems. In fact, the psychomotor test did not show that the motor capacity of the treated animals was impaired. These are amazing results because literature indicates that neonatal or chronic hypothyroidism provokes severe neurological defects. Nevertheless, previous studies [18,21] have not indicated any serious defects in animals treated perinatally with methimazole in their drinking water. These results suggest that this treatment, despite being chronic, could be considered mild. Chronic hypothyroidism does not seem to affect activity. The increased ambulation of the hypothyroid group in the Boissier test was probably more related to the exploration of the new environment rather than to activity. In fact, no increase in ambulation was observed on the second day of the open-field test. As stated earlier in Section 2, measures from the second day of this test are less contaminated by exploration. Our results indicate that the hypothyroid animals in the open-field test ambulated more on the first day, i.e. when it was a novel environment, whereas the hyperthyroid animals ambulated more on the second day, i.e. when it was a known environment. These results suggest that the characteristic hyperactivity of rehabilitated animals from congenital or perinatal hypothyroidism [11,12,19,21,26 – 28,31] does not appear if the hypothyroid state continues. In previous studies, we have observed that adult hypothyroidism does not affect activity
either [22,23]. Our present data differs from the results of another study [13] in which animals were radiothyroidectomized 4 h after delivery. In this study, it was found that chronic hypothyroidism increased locomotor activity, but there was no normal increase in deaths or illness in severe treatments reported, so it is not unlikely that certain compensatory processes have taken place. It has also been reported that hypothyroidism affects the activity circadian rhythm, but the administration of thyroxine to untreated animals does not [29]. Therefore, it seems that the control of the circadian effects could be relevant in hypothyroid animals. This suggests that the absence of any effects of hypothyroidism on activity in our experiment could be influenced by the moment at which the animals were tested. In fact, Schull et al. [30] showed that the activity levels of thyro-parathyroidectomized animals increase more abruptly as it gets dark than sham-operated did. Nevertheless, as mentioned earlier, the administration of thyroxine does not affect the activity circadian rhythm, so it seems that the increase in the activity of the hyperthyroid group observed in our experiment is not related to the activity circadian rhythm. Different biological substrates could be implicated in thyroid hormones effect on activity. In fact, hypothyroidism depresses dopaminergic [10,32 – 35] and noradrenergic [10,36,37] activity. There is considerable evidence to indicate that novelty-seeking behaviour involves the activation of the mesolimbic DA system. It appears that D1 receptors may mediate the incentive rewarding properties of novelty, whereas D2 receptors may be more important in mediating locomotor performance per se [38]. It will be interesting to delimit whether the effects observed for hypo- and hyperthyroidism are the result of the thyroid action over D1 or D2 receptors. The perinatal period seems extremely vulnerable to hypothyroidism with respect to changes in locomotor activity. In addition, chronic but not acute dysthyroidism modifies utilization of dopamine [39,40]. Hypothyroidism induces the loss of dopamine receptors in the developing brain [34], which might sensitise them. If this sensitisation is permanent, continued hypothyroidism could counteract the effects of chronic dysthyroidism on the dopaminergic system. This hypothesis would account for the lack of effects on activity observed in chronic hypothyroid animals. However, if normal thyroid levels are recovered, and therefore the activity of the noradrenergic and dopaminergic systems too, the permanent sensitisation of the dopamine receptors would lead to an increase in the functionality of these systems. This could explain the increase in activity in animals recovered from perinatal hypothyroidism [11,12, 19,21,26 – 28,31], some effects of the therapies [4,5] and the hyperactivity in the thyroid hormone resistance syndrome [7,8]. At the beginning of the behavioural assessment, motor capacity and basic reflexes were evaluated so that impaired motor capacity could be rejected as a causal factor of the results obtained in the activity and exploration tests. The
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results in the psychomotor battery confirmed that the basic reflexes analysed were correctly developed and the motor capacity of the animals was not impaired. In fact, hypothyroidism delays and hyperthyroidism accelerates the appearance of these reflexes, but both groups of animals eventually develop them normally [41]. Interestingly, the hypothyroid group performed better in this battery than both the control and the hyperthyroid groups. Namely, they remained longer on the inclined screen test and turned more quickly in the negative geotaxis test. This better performance could well be related to behavioural differences in coping with situations that are potentially anxiogenic because of the danger of falling or novelty. In fact, we found that perinatal and chronic hypothyroidism induced an anxiolytic behavioural pattern measured by the plus maze in adulthood [18,21,22]. Our results showed that chronic hyperthyroidism increases activity. As far as we know, there is no data on the effects of chronic hyperthyroidism on activity from the perinatal period to adulthood. Neonatal hyperthyroidism seems to increase activity [42], but this hyperactivity does not continue into adulthood when animals recover their normal state [21,43]. However, it has been pointed out that single intraperitoneal injections of high dosages of thyroid hormones in the neonatal period cause hyperactivity in adulthood [44 –46]. Adult hyperthyroidism also seems to produce an increase in activity [22,36,42], although there are discrepant results [10]. The increase in dopamine [42,47] or noradrenaline [42,48] activity is also believed to be the mechanism implicated in the hyperactivity caused by hyperthyroidism. However, in this case, the effect could be more activational (i.e. dependent on current hormone levels) than the organizational effects observed in hypothyroidism. Nevertheless, as mentioned earlier, high dosages of T4 or T3 on the third or fourth day after delivery provokes permanent hyperactive behaviour [44 –46]. It is therefore likely that there are short periods of time, in which the organism is highly sensitive to variations in levels of thyroid hormone, and these could affect the organization of the pituitary – thyroid axis. Nevertheless, this effect may well be more closely related to the high dosages of thyroid hormones than to the affected period. Related to exploration behaviour, the results obtained with the light– darkness test suggest that the discriminant function measured static exploration by considering the fact that variables such as glances, the number of head dippings and the time spent on the illuminated side had a positive influence on the function, while the number of compartmental changes influenced it negatively. The hypothyroid animals explored more than the control and hyperthyroid animals, although they were not as active as the hyperthyroid animals. In fact, as stated above, hypothyroid animals ambulated more on the open field during the first day than on the second day. The decrease in ambulation on the second day could be related to familiarity with the
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environment. A similar effect in the light– darkness test was observed in adult animals that had recovered from perinatal hypothyroidism [21], but, in this test, adult hypothyroid animals did not explore more than the control animals did [22]. This data suggests that the effect on exploration would be an organizational effect, limited to the perinatal period. This could be related to the anxiolytic behavioural pattern observed in perinatal hypothyroidism [21]. Brucker-Davis [17] after an exhaustive revision indicated that several categories of synthetic chemicals are suspected to be thyroid disruptors or modulators, potentially altering the thyroid economy at multiple levels. In fact, data available from two accidental polychlorinated biphenyl (PCB) poisonings in Japan and in Taiwan indicate that neurodevelopmental deficit, behaviour and cutaneous signs were the dominant clinical features. It is interesting that Yucheng patients (from a Taiwan accident), who presented with an increase of free thyroxine and T3, were described as hyperactive, which is coincident with the results of our study, and with the hypothesis that the attention deficithyperactivity disorder could be related to hyperthyroidism. All human studies at different levels of exposure and contamination show some degree of neurodevelopment impairment. However, the impact on humans may depend on timing, dose and duration of exposure, synergy between chemicals, as well as genetic and immune status predisposing the exposed person to thyroid diseases [17]. In conclusion, the results of this experiment indicate that chronic thyroid deficiency increases exploration but not activity. In contrast, chronic hyperthyroidism increases activity but not exploration. These results agree with the hypothesized effects of chronic dysthyroidism on exploratory behaviour. The hypothesized effects on activity behaviour were only confirmed in the hyperthyroid group, whereas no differences in this behaviour were observed in the hypothyroid group. This suggests that the effects of perinatal hypothyroidism in activity could be counteracted by the effects of adult hypothyroidism. Acknowledgements We gratefully acknowledge Eva Estebanez’s collaboration and Dr. Dolors Ribas’ advice in our statistical analysis. References [1] Rochiccioli P, Roge´ B, Alexandre F, Tauber MT. School achievement in children with hypothyroidism detected at birth and search for predictive factors. Horm Res 1992;38:236 – 40. [2] Gottschalk B, Richman RA, Lewandowski L. Subtle speech and motor deficits of children with congenital hypothyroid treated early. Dev Med Child Neurol 1994;36:216 – 20. [3] Derksen-Lubsen G, Verkerk PH. Neuropsychologic development in early treated congenital hyperthyroidism: analysis of literature data. Pediatr Res 1996;39(3):561 – 6.
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