Behavioral inhibition and impaired spatial learning and memory in hypothyroid mice lacking thyroid hormone receptor α

Abstract

Thyroid hormone insufficiency leads to impaired neurogenesis, behavioral alterations and cognitive deficits. Thyroid hormone receptors, expressed in brain regions involved in these behaviors, mediate the effects of thyroid hormone deficiency or excess. To determine the contribution of thyroid hormone receptor alpha (TRα) in these behaviors, we examined the behavior of euthyroid as well as hypo- and hyperthyroid mice lacking all isoforms of the TRα (TRα^o/o). The hypothyroxinemic TRα^o/o mice demonstrated behavioral inhibition, manifested in decreased activity and increased anxiety/fear in the open field test (OFT) and increased immobility in the forced swim test (FST) compared to C57BL/6J mice. TRα^o/o mice also showed learning and recall impairments in the Morris water maze (MWM), which were exaggerated by hypothyroidism in TRα^o/o mice. These impairments were concurrent with increased thigmotaxis, suggesting an increased anxiety-like state of the TRα^o/o mice in the MWM. Expression of genes, known to be involved in processes modulating learning and memory, such as glucocorticoid receptor (GR), growth-associated protein 43 (GAP-43) and neurogranin (RC3), were significantly decreased in the hippocampus of TRα^o/o mice. GR expression was also decreased in the frontal cortex and amygdala of TRα^o/o mice, indicating that expression of GR is regulated, probably developmentally, by one or more isoforms of TRα in the mouse brain. Taken together these data demonstrate behavioral alterations in the TRα^o/o mice, indicating the functional role of TRα, and a delicate interaction between TRα and TRβ-regulated genes in these behaviors. Thyroid hormone-regulated genes potentially responsible for the learning deficit found in TRα^o/o mice include GR, RC3 and GAP-43.

Introduction

In all vertebrates, thyroid hormones are essential for normal brain development [11], [12], [27], [61]. Experimentally induced moderate and transient deficiency of maternal thyroid hormones (TH) have profound effects on fetal neurogenesis [5] and brain growth; even mild hypothyroidism of the mother decreases maternal thyroxine (T4) transport to the fetus [57]. This mild maternal hypothyroidism can negatively affect the child's neuropsychological development [20], [42], since the fetal thyroid is unable to produce any T4 before 12–14 weeks of gestation [42].

Research suggests that thyroid function abnormalities in adulthood can also have profound behavioral consequences. Clinical hypothyroidism is often associated with frank neuropsychological and affective alterations, and considered to be one of the causes of reversible dementia. Even mild hypothyroidism predicts lower levels of memory performance [55]. Additionally, moderate to severe depression and mania are associated with reduced TSH response to TRH [44].

In animals, hyperthyroidism in adulthood leads to increased activity in the open field test [46], [50], and both experimental hypo- [25] and hyperthyroidism [46] have been reported to increase immobility behavior in the forced swim test. Spatial learning and memory deficits have been found in the genetically hypothyroid hyt/hyt mouse [3], and in congenitally hypothyroid animals [1], [2], [32].

Behavioral consequences of developmental or adult thyroid dysfunction are mediated by actions of thyroid hormones on thyroid hormone receptors (TR), predominantly by positive or negative transcriptional regulation of thyroid hormone responsive genes [39]. The ligand-activated nuclear TR isoforms, TRα1, TRβ1 and TRβ2, are produced by alternative splicing of the primary transcripts of the TRα and TRβ genes, respectively [52], [58]. Another spliced product of the TRα gene, TRα2, does not bind TH [29]. The functions of the TRΔα1 and TRΔα2 isoforms, which are also expressed in the brain, are still unknown [31]. Recently, mice, lacking both TRα1 and TRα2 and the shorter transcripts initiated from the internal promoter located in intron 7 [15] have been generated offering the opportunity to characterize the role of these receptors in various behaviors. These TRα^o/o mice have decreased T4, but normal TSH levels [16], [31]. As such, the TRα^o/o mice can model the mild hypothyroxemia, which leads to behavioral consequences, when it occurs during development and also in adulthood.

In this study, we aimed to determine the behavioral consequences of deletion of the TRα locus. To achieve this aim we employed TRα^o/o mice and wild type C57BL/6J (B6) mice (the genetic background of the TRα^o/o mice). Since we preferred to assure the normal thyroid milieu of the fetal C57BL/6J mice, we did not choose wild type littermates of the TRα^o/o mice as controls. Instead, we considered the TRα^o/o and C57BL/6J mice as two separate strains that differ genetically at their TRα locus, and developmentally in their thyroid hormonal milieu. To evaluate the role of the adult hypothyroxinemia on behaviors known to be affected by thyroid hormones, we induced hypo- or hyperthyroidism in both strains of adult mice. The behaviors examined included spatial learning and memory in the Morris water maze (MWM), activity and anxiety/fear in the open field test (OFT) and immobility in the forced swim test (FST). In addition, we evaluated the expression of GAP-43 and glucocorticoid receptor (GR), both implicated in learning [38], [48], [60], and the thyroid hormone-regulated gene, neurogranin (RC3), in brain regions involved in these behaviors.