Thyroxine (T4) in neuronal tissue, Decreased

Thyroid hormones (TH) are present in brain tissue of most vertebrate species, and thyroxine (T4) is converted to triiodothyronine (T3) locally in this tissue.  The amount of THs in brain is known to vary during development and to differ among brain regions (Calvo et al., 1990; Kester et al., 2004; Tu et al., 1999). In human cerebral cortex, T3 increases steadily from 13-weeks, reaching adult levels by 20 weeks post conception. This occurs despite very low and unchanging levels in fetal serum T3, when fetal serum T4 increases 3-fold over the same period. This indicates that T3 in fetal brain is locally generated from serum-derived T4 via the activity of deiodinases, primarily DIO2. DIO2 serves to convert T4 to T3. During this time in fetal development DIO3 activity, which converts T3 to the inactive reverse T3 (rT3), remains very low in cortex.  In contrast, in other brain regions including hippocampus and cerebellum, T3 remains low throughout early and mid-gestation and corresponds with high activity of DIO3 in these brain regions. In late gestation and after birth, DIO3 levels drop in hippocampus and cerebellum with a corresponding increase in T3 concentrations (Kester et al., 2004). 

A similar spatial and temporal profile of deiodinase activity and corresponding brain hormone concentrations has been observed in rodent brain (Calvo et al., 1990; Tu et al., 1999). In the rat, either whole brain or cortex have been preferentially assessed due to the low levels of hormones present and the small tissue volumes make quantitification difficult. Brain T3 and T4 rise in parallel from gestational day 10 to gestational day 20 in rat. They are typically both quite low until gestational 17 with steep increases between GD18 and GD20 corresponding to the onset of fetal thyroid function (Calvo et al., 1990; Ruiz de Ono et al., 1988; Obergon et al., 1981). Just before birth, brain T3 and T4 concentrations are about one-third to one-half that of adult brain. Brain development in the early postnatal period in rat is roughly equivalent to the 3^rd trimester in humans such that adult levels of T3 and T4 in brain are not reached in rodents until the 2^nd-3^rd postnatal week.

For THs to gain access to brain tissue they need to cross the blood brain barrier (BBB) which regulates the active transport of TH into neurons. Many transporter proteins have been identified, and the monocarboxylate transporters (Mct8, Mct10) and anion-transporting polypeptide (OATP1c1) show the highest degree of affinity towards TH and are prevalent in brain (Jansen et al., 2007; Mayer et al., 2014).  Transporters express a distinct distribution pattern that varies by tissue and age (Friesema et al., 2005; Henneman et al., 2001; Visser et al., 2007; Heuer et al., 2005; Muller and Heuer, 2007). Although several transporters have been identified, current knowledge of cell specific profile of transporters is limited. 

Most of the hormone transported across the blood brain barrier is in the form of T4, primarily through the cellular membrane transporters (e.g., OATP1c1 transporter) into the astrocyte (Visser and Visser, 2012; Sugiyama et al., 2003; Tohyama et al., 2004). Within the astrocyte, T4 is converted into T3 via the local activity of deiodinase 2 (DIO2) (Guadano-Ferraz et al., 1997).  A small amount of T3 may cross the blood brain barrier directly via the T3-specific transporter, MCT8 (Heuer et al., 2005). Although in mature brain T3 derives partially from the circulation and from the deiodination of T4, in the fetal brain T3 is exclusively a product of T4 deiodination (Calvo et al., 1990; Grijota-Martinez et al., 2011). In both cases, only the required amount of T3 is utilized in neurons and the excess is degraded by the neuron-specific deiodinase DIO3 (Tu et al., 1999; St. Germain et al., 2009; Hernandez et al., 2010).

Both deiodinase and transporter expression in brain peak in different brain regions at different times in fetal and neonatal life (Kester et al., 2004; Bates et al., 1999; Muller and Heuer, 2014; Heuer, 2007). Collectively, these spatial and temporal patterns of transporter expression and deiodinase activity provide exquisite control of brain T3 available for nuclear receptor activation and regulated gene expression.

Radioimmunoassays (RIAs) are commonly used to detect TH in the brain (e.g., Obregon et al., 1982; Calvo et al., 1990; Morse et al., 1996; Bansal et al., 2005; Gilbert et al., 2013). The method (and minor variants) is well established in the published literature. However, it is not available in a simple 'kit' and requires technical knowledge of RIAs, thus has not been used in most routine toxicology studies. Evaluations in neuronal tissue are complicated by the difficulty of the fatty matrix, heterogeneity of regions within the brain, and low tissue concentrations and small tissue amounts especially in immature brain. Most often whole brain homogenates are assessed, obfuscating the known temporal and regional differences in brain hormone present. Two analytical techniques, LC- and HPLC-inductively coupled plasma–mass spectrometry have recently been used to measure brain concentrations of TH. These techniques have proven capable of measuring very low levels in whole-body homogenates of frog tadpoles at different developmental stages (e.g., Simon et al., 2002; Tietge et al., 2010). The assay detects I–, MIT, DIT, T4, T3, and rT3. More recently, Wang and Stapleton (2010) and Donzelli et al. (2016) used liquid chromatography-tandem mass spectrometry for the simultaneous analysis of five THs including thyroxine (T4), 3,3′,5-triidothyronine (T3), 3,3′,5′-triiodothyronine (rT3; reverse T3), 3,3′-diiodothyronine (3,3′-T2), and 3,5-diiodothyronine (3,5-T2) in serum and a variety of tissues including brain. These analytical methods require expensive equipment and technical expertise and as such are not routinely used.

THs are critical for normal brain development in most vertebrates, primarily documented empirically in mammalian species (Bernal, 2013).  However, there is compelling data that demonstrates the need for TH in brain development for many other taxa, including: birds, fish and frogs (Van Herck et al., 2013; Denver, 1998; Power et al., 2001). The most well known non-mammalian action of TH is to induce metamorphosis in amphibians and some fish species. However, there is a fundamental difference in the mechanisms by which T3 affects amphibian metamorphosis vs its role in mammalian brain development (Galton, 1983). In the rat, brain development proceeds, even if defective, despite the absence of TH. By contrast, TH administration to tadpoles induces early metamorphosis, whereas in its absence, tadpoles grow to extremely large size, but the metamorphosis program is never activated (Galton, 1983).

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