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Experimental Physiology – Research Paper
Perinatal and chronic hypothyroidism impair behavioural development in male and female rats N. van Wijk, E. Rijntjes and B. J. M. van de Heijning Human and Animal Physiology group, Department of Animal Sciences, Wageningen University, PO Box 338, 6700 AH, Wageningen, The Netherlands
A lack of thyroid hormone, i.e. hypothyroidism, during early development results in multiple morphological and functional alterations in the developing brain. In the present study, behavioural effects of perinatal and chronic hypothyroidism were assessed during development in both male and female offspring of hypothyroid rats. To induce hypothyroidism, dams and offspring were fed an iodide-poor diet and drinking water with 0.75% sodium perchlorate; dams starting 2 weeks prior to mating and pups either until the day of killing (chronic hypothyroidism) or only until weaning (perinatal hypothyroidism) to test for reversibility of the effects observed. Neuromotor competence, locomotor activity and cognitive function were monitored in the offspring until postnatal day 71 and were compared with age-matched control rats. Early neuromotor competence, as assessed in the grip test and balance beam test, was impaired by both chronic and perinatal hypothyroidism. The open field test, assessing locomotor activity, revealed hyperactive locomotor behavioural patterns in chronic hypothyroid animals only. The Morris water maze test, used to assess cognitive performance, showed that chronic hypothyroidism affected spatial memory in a negative manner. In contrast, perinatal hypothyroidism was found to impair spatial memory in female rats only. In general, the effects of chronic hypothyroidism on development were more pronounced than the effects of perinatal hypothyroidism, suggesting the early effects of hypothyroidism on functional alterations of the developing brain to be partly reversible and to depend on developmental timing of the deficiency. (Received 21 February 2008; accepted after revision 16 June 2008; first published online 20 June 2008) Corresponding author N. van Wijk: Danone Research, PO Box 7005, 6700 CA, Wageningen, The Netherlands. Email:
[email protected]
Some of the most prominent actions of thyroid hormones occur during fetal and postnatal brain development. The symptoms of hypothyroidism during early development (e.g. growth impairment and multiple morphological and functional alterations) reflect a maternal, fetal or postnatal thyroid hormone deficiency (Boyages & Halpern, 1993; Porterfield & Hendrich, 1993; Forrest, 2004; Zoeller & Rovet, 2004). The severity of the effects depends on the magnitude, the time of onset and the onset of treatment of the deficiency (Porterfield & Hendrich, 1993). Thyroid hormones affect several processes in brain development during different time windows, e.g. neuronal and glial cell differentiation and proliferation, axonal and dendritic growth, synapse formation, cell migration and myelination (Thompson & Potter, 2000; Anderson, 2001; Bernal, 2002; Zoeller & Rovet, 2004; Huang et al. 2008). This eventually results in anatomical alterations in the cerebellum, cerebrum, hippocampus and other C 2008 The Authors. Journal compilation C 2008 The Physiological Society
brain structures (Dussault & Ruel, 1987; Porterfield & Hendrich, 1993; Thompson & Potter, 2000; Anderson, 2001; Bernal, 2002; Smith et al. 2002; Zoeller & Rovet, 2004). Furthermore, several neurotransmitter systems that play a functional role in activity, mood and learning, hence in behaviour, such as the GABAergic, cholinergic, dopaminergic and serotonergic systems, are altered in hypothyroid rats (Rastogi & Singhai, 1979; Dussault & Ruel, 1987; Darbra et al. 1995; Evans et al. 1999; Friedhoff et al. 2000; Sala-Roca et al. 2002a; Smith et al. 2002; Broedel et al. 2003; Ortiz-Butron et al. 2003; Auso´ et al. 2004; Friedhoff et al. 2000). Behavioural alterations resulting from prenatal, perinatal, postnatal, chronic and adult-onset hypothyroidism have been studied in depth in both man and animals. Human prospective and retrospective studies focused primarily on cognitive functions, mood disorders and neuromotor capabilities. Many rat studies DOI: 10.1113/expphysiol.2008.042416
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on hypothyroidism evaluated the effects on cognitive functions, locomotor activity and neuromotor competence (Hendrich et al. 1984; Tamasy et al. 1986; Akaike et al. 1991; Darbra et al. 1995, 2003, 2004; Goldey et al. 1995; Friedhoff et al. 2000; MacNabb et al. 2000; Brosvic et al. 2002; Sala-Roca et al. 2002b; Negishi et al. 2005; Dos Reis-Lunardelli et al. 2007; Huang et al. 2008). Comparisons reveal contradictory results, mainly attributable to differences in severity (i.e. treatment method) and developmental timing of the deficiency (i.e. the treatment period). Propylthiouracil and methimazole have been intensively used in animals to induce hypothyroidism, since these agents affect thyroid hormone synthesis and deiodinase activity directly. However, propylthiouracil and methimazole appear to have general toxic effects (Zoeller & Crofton, 2005). In the present study, an alternative method to induce hypothyroidism during development was employed, whereby dams were fed a diet virtually devoid of iodide, starting 2 weeks before pregnancy. In addition, sodium perchlorate (0.75%) was added to their drinking water in order to stimulate iodide wasting by blocking the (re-)uptake of iodide in the thyroid gland and kidney (Wolff, 2001). In this way, a gradual iodide deficiency-based hypothyroid status was imposed onto the animals. As a consequence, both dams and fetuses were hypothyroid during the entire period of pregnancy and thereafter. The reversibility of the effects of hypothyroidism on development was tested in a group in which hypothyroidism was present only perinatally, until postnatal day 14. Postnatal day 14 was chosen because the critical period of the developmental actions of thyroid hormones is confined to 2–3 weeks postpartum (Dussault & Ruel, 1987; Porterfield & Hendrich, 1993; Thompson & Potter, 2000; Clancy et al. 2001; Kobayashi et al. 2005). Thus, the effects of both perinatal and chronic hypothyroidism on the behavioural and physical development in the male and female offspring were assessed in the present study.
Exp Physiol 93.11 pp 1199–1209
offspring. Health status was checked daily, and animals were weighed weekly. All experimental procedures were approved by the Wageningen University Animal Ethics committee. Two weeks prior to mating, the female breeding animals were divided into two experimental groups. In one group, hypothyroidism was induced; the other group served as a control group. Hypothyroidism was induced by a ‘hypothyroid’ diet. The hypothyroid animals received an iodide-poor (i.e. without added iodide) pelleted food (Research Diet Services, Wijk bij Duurstede, The Netherlands) and Millipore-filtered water with 7.5 g l−1 sodium perchlorate (NaClO 4 .H 2 O, Merck, Darmstadt, Germany). The control group received pelleted food (Research Diet Services) with normal levels of iodide (0.2 mg iodide kg−1 ) and normal tap water. The pellet composition was according to AIN-93G guidelines (Reeves et al. 1993), with or without iodide. Food and water intake did not differ in dams that were fed either the hypothyroid diet or the control diet. All 12 dams gave birth on the same day; the day of birth was designated as postnatal day 1. Four days after birth, the litters were sexed and subsequently divided between six foster mothers (4 hypothyroid dams and 2 control dams). Each litter contained eight pups: four males and four females to exclude possible effects of litter size and composition on the development of the pups. Spare pups were killed by decapitation. From postnatal day 14 onward, two hypothyroid dams received the control diet: the ‘hypo-normal’ group. Thus three treatment groups were formed for both sexes: a control group, a hypothyroid group and a hypo-normal group, each consisting of two dams with eight pups each. The pups were weaned and separated from the dams at postnatal day 29. The pups were then group housed (4 females or 4 males). Each cage contained two pairs of siblings obtained from two different litters. From postnatal day 48 onward, the rats were housed in pairs; littermates were separated. All test animals were killed on postnatal day 76 by CO 2 –O 2 inhalation (2.0 l min−1 CO 2 and 1.0 l min−1 O 2 ). The dams were killed after weaning (on postnatal day 29).
Methods Animals and experimental procedures
The experiments were conducted with 24 female and 24 male Wistar (HsdCpb:WU) rats derived from 12 breeding pairs purchased from Harlan (Horst, The Netherlands). The 12 dams were obtained from a pool of 67 dams from concurrent experiments. Temperature and relative humidity were kept constant at 22 ± 1◦ C and 55 ± 10%, respectively. Rats were maintained on a 10 h–14 h light–dark cycle with lights on at 03.00 h (light intensity, approx. 80 lx). Experimental diets (water and food) were available ad libitum to the dams and their
Body temperature
Body temperature was monitored by means of a transponder–scanner system (IPTT-100 / Pocket Scanner, Bio Medic Data Systems Inc., Seaford, DE, USA) to determine differences in thermoregulation and metabolism between the experimental groups. Therefore, on postnatal day 27 all male rats received a subcutaneous transponder in the neck region. The transponders were implanted under light isoflurane (IsoFloTM , Abbott Laboratories Ltd, UK) anaesthesia (3% isoflurane C 2008 The Authors. Journal compilation C 2008 The Physiological Society
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Hypothyroidism and behavioural development
vaporized in a mixture of 1.0 l min−1 O 2 and 1.0 l min−1 N 2 O) with a syringe-like action using a hollow needle. Body temperature of undisturbed animals was thus measured frequently with a transponder scanner between 08.00 and 17.00 h. Owing to the limited number of transponders available, body temperature was monitored in male rats only. Radioimmunoassay (RIA)
To determine thyroid status, blood samples were collected from the pups, and from dams randomly selected from the pool of 67 dams. Samples from the dams were obtained 1–4 days before conception (i.e. 2 weeks after the start of the experimental diets) and 2–5 days after delivery, by a tail-nick procedure (Fluttert et al. 2000). At the time of killing of dams (postnatal day 29) and pups (postnatal day 76), blood samples were collected by cardiac puncture. The collected blood samples were centrifuged for 5 min at 8 400 g. The plasma was collected and stored at −20◦ C. Total plasma thyroid-stimulating hormone (TSH), thyroxine (T 4 ) and 3,5,3 -triiodothyronine (T 3 ) concentrations were determined by RIA. The concentration of TSH was determined with NIDDK-antirat TSH-RIA-6 antibody and NIDDK-rTSH-I-9 tracer (National Institute of Diabetes, Digestive and Kidney Diseases, Bethesda, MD, USA). The tracer was labelled with 125 I (GE Healthcare, Amersham, UK). NIDDKrTSH-RP-3 was used for the standard curve (sensitivity of the assay, 0.2 ng ml−1 ; inter- and intra-assay variance,
