**5. Explanations of thyroid hormone effects on the brain at the molecular and cellular level**

Owing to the complex actions of thyroid hormone there is currently no concluding explanation concerning the molecular mechanisms underlying the effects of thyroid hormone on cortical excitability. At the morphological level, in the mature brain thyroid hormone excess (149) as well as deficiency (150) have been reported to decrease the number of dendritic spines, assumed to represent postsynaptic endings, already after 5 days in adult rats. Even more dramatically, hypothyroidism leads to a reduction of the neuropile in CA1 and CA3 hippocampal areas and in addition to a loss of pyramidal cells in the CA1 area (151). Thus it seems possible that adult-onset hypothyroidism may actually cause neuronal degeneration, as already occasionally observed in autopsies of early cases of patients who had died with myxedema (152, 153). A reversible shrinkage of neuropile could also explain the findings of reversibly widened ventricular spaces in the brains of hypothyroid subjects (154, 155).

The development of cholinergic terminals in rat forebrain, hippocampus and amygdala is regulated to a considerable extent by thyroid hormone (see e.g. (156)). Although smaller and more localized effects are reported in adults, several lines of evidence suggest that acetylcholine -release may be enhanced by thyroid hormone and decreased in hypothyroidism in the adult nervous system as well (157, 158). A decrease of cholinergic activity could perhaps also explain the occurrence of slow EEG waves (159) as well as the cognitive impairments frequently seen in hypothyroid subjects. A regulation of cholinergic function also fits to the observation of a regulation of nerve growth factor which has been suggested to be involved in maintaining the function of cholinergic hippocampal projections by thyroid hormone in adult rat brain (160). Thyroid hormone, however, does not seem to interfere exclusively with cholinergic forebrain neurons but to regulate the balance of a variety of other neurotransmitters in a region-specific manner. Hence dopamine levels were found to be increased in the midbrain of hyperthyroid rats (161) and decreased in hypothyroid rats (162). Also the dopaminergic input into striatal neurons could be upregulated by thyroid hormone (163). Furthermore, a differential regulation of serotonin levels (162, 164) as well as 5-HT2 receptors have been found (165). Regulations of various adrenoceptors as well as GABAreceptors have been described see e.g. (166–168). In addition T3 could act as a cotransmitter to modulate noradrenergic action (169) or as a modulator of endogenous benzodiazepine action (170). While it is believed that thyroid hormone exerts its effects predominantly via nuclear receptors possible direct effects on membrane receptors further complicate the picture (157, 171, 172). In addition to a membrane action via αVβ3intergins, high doses of 20 µM T3 or T4 have been shown to directly act on GABA receptors to down-regulate GABAergic postsynaptic currents in cultured hippocampal neurons (173, 174), which could explain acute increases in neuronal excitability induced by iontophoretecally injected T4 and T3 (171). Although the regulatory influences exerted by thyroid hormone are complex it seems that T3 regulates to some extent the release of neurotransmitters such as acetylcholine, dopamine, 5-HT and noradrenalin in specific pathways as well as the density of the corresponding receptors (166).

102 Thyroid Hormone

in the adult.

**and cellular level** 

Taken together, the auditory system may lose its sensitivity to thyroid hormone with increasing age and this may also depend on an individual susceptibility. In addition, effects on hearing may develop only after a thyroid hormone withdrawal for more than five weeks

The present tests performed on a small number of patients indicate that the most prominent symptom after 4 weeks of thyroid hormone withdrawal is a beginning decline in the speed of central neuronal information processing, which was reflected in decreases in the speed of visual perception, speed of speech as well as of visual-spatial orientation. Hearing and smelling thresholds were only slightly changed, and in the context with the publications discussed above this indicates that auditory and olfactory perception may change only with thyroid dysfunctions of longer duration or are more sensitive to thyroid hormone in development. The experiments illustrated here complement previous findings, that hypothyroidism slows peripheral conduction velocity (144), reduces EEG frequencies and increases latencies of evoked potentials (73). The conception that thyroid hormone deficiency causes a general decrease in neuronal excitability was recently supported by the observation of a decreased cortical excitability and increased motor thresholds using transcranial magnetic stimulation in adult patients (145). Accordingly, in a small percentage of epileptic seizures in humans (146) thyrotoxicosis was identified as sole cause of the seizures and the seizures were found to fully subside after restoration of euthyroidism, again indicating an effect of thyroid hormone on cortical excitability. An increased susceptibility to seizures was

*Effects of thyroid hormone on sensory perception and brain function*

also noticed in hyperthyroid animals such as cats (147) and mice (148).

**5. Explanations of thyroid hormone effects on the brain at the molecular** 

Owing to the complex actions of thyroid hormone there is currently no concluding explanation concerning the molecular mechanisms underlying the effects of thyroid hormone on cortical excitability. At the morphological level, in the mature brain thyroid hormone excess (149) as well as deficiency (150) have been reported to decrease the number of dendritic spines, assumed to represent postsynaptic endings, already after 5 days in adult rats. Even more dramatically, hypothyroidism leads to a reduction of the neuropile in CA1 and CA3 hippocampal areas and in addition to a loss of pyramidal cells in the CA1 area (151). Thus it seems possible that adult-onset hypothyroidism may actually cause neuronal degeneration, as already occasionally observed in autopsies of early cases of patients who had died with myxedema (152, 153). A reversible shrinkage of neuropile could also explain the findings of

reversibly widened ventricular spaces in the brains of hypothyroid subjects (154, 155).

The development of cholinergic terminals in rat forebrain, hippocampus and amygdala is regulated to a considerable extent by thyroid hormone (see e.g. (156)). Although smaller and more localized effects are reported in adults, several lines of evidence suggest that acetylcholine -release may be enhanced by thyroid hormone and decreased in hypothyroidism in the adult nervous system as well (157, 158). A decrease of cholinergic activity could perhaps A stimulating effect of thyroid hormone on transmitter synthetic enzymes or precursoruptake systems as well as the protein synthesis of the receptors could in principle explain the decrease in cerebral responsiveness in hypothyroid subjects. Furthermore, a downregulation of postsynaptic inhibitory currents in hyperthyroidism, as suggested by Puia and Losi (174), could account for the increased irritability seen in hyperthyroid subjects. A diminished postsynaptic current density due to a decrease in transmitter release or receptor density or activation could also explain some of the increased latencies since a smaller current density would lead to a delay in the charging of the membrane capacitance. However, investigations using transcranial magnetic stimulation provided evidence that in hypothyroid patients the cortical excitability as such is decreased (145). Furthermore, experiments on peripheral nerves of hyperthyroid rats indicated enhanced afferent spikes and a drop in the chronaxia for direct activation of action potentials in rat peripheral nerves (175). Hence thyroid hormone could also influence neuronal excitability directly, which could secondarily result in a decrease in transmitter release.

*Changes in conduction velocity, action potential waveform and the regulation of voltage-gated ion currents by thyroid hormone*

Changes in Achilles tendon reflexes and the slowing of peripheral conduction velocity in hypothyroidism have so far mostly been explained by a reduction in myelination, and the gene for myelin basic protein is, in fact, regarded as one of the few genes known to be directly regulated by thyroid hormone (for review see (176)). However, a decrease in

sodium current density could as well explain the decreases in peripheral conduction velocities and increases in latencies of evoked potentials found in hypothyroidism (16–24) and reversely the increased amplitudes in hyperthyroidism (177, 178). Since there seems to be an optimal density of sodium channels that ensures maximal neuronal conduction velocity (179), beyond which no further increase or even a slowing of conduction velocity occurs, an upregulation of sodium currents by thyroid hormone could also explain the inconsistent findings concerning latencies of evoked visual potentials in hyperthyroidism, where some authors found decreases in latencies (180) or even increases with increases in thyroid hormone (21, 71, 177, 178). Because of the temperature sensitivity of the activation of sodium and calcium currents the fall in core temperature during hypothyroidism and its increase in hyperthyroidism could further exacerbate the symptoms (19).

Thyroid Hormone Effects on Sensory Perception, Mental Speed, Neuronal Excitability and Ion Channel Regulation 105

gated Na+-currents (INav) by T3 (192). An upregulation of the density of voltage-gated Na+ currents was also found in acutely isolated neurons from the occipital cortex of hyperthyroid rats and a down regulation observed in cells from hypothyroid rats. The changes in Na+current density led to increased action potential upstroke velocities as well as to enhanced discharge rates in thyroid hormone treated cells in response to identical stimulus strengths (193). Similarly, increases in voltage-gated Na+ currents were observed in human neuroepithelial cells as well as mesenchymal stem cells after incubation with 1 nM T3 for 72h to 6 days in culture. Neuroepithelial cells additionally responded with increases in Ca2+ currents to prolonged T3-treatment (194). In the in *vivo* situation thyroid hormone effects seem to be more complex: Thus in CA1 neurons of the rat hippocampus changes in the bursting pattern have been observed, which could be explained by an upregulation of a low-threshold Ca2+ current (195). Furthermore, consistent with Hoffmann and Dietzel, 2004, decreases in action potential depolarization rate and decreases in discharge rate were observed by thyroid hormone withdrawal. In contrast to the action of thyroid hormone in the heart these authors additionally observed a shortening of action potental duration upon thyroid hormone withdrawal, that could be explained by the upregulation of an A-type potassium current (196). Finally in a somewhat distant animal, in Rohon-Beard neurons from the embryonic zebrafish rapid increases of voltage-gated Na+currents by thyroxine were found (197). This Na+current regulation was shown to be essential for the further development of the embryo and depended on αVβ3 integrin activation and the MAPK (p38) pathway (198). An increase in voltage-gated Na+current density by thyroid hormone would

cause a general speeding of mental functions as illustrated in figure 5:

**Figure 5.** Simulation of action potential spread in a hippocampal model neuron, for action potential with high (A-red) and low (A-blue) Na+-current density. A an increase in Na+current density increases action potential depolarization (Aa), amplitude and discharge frequency (Ab). B. Simulation of action

potential spread in a ramified neuron 1 ms after application of a stimulus at t=1ms.

#### *Influences of thyroid hormone on action potentials and underlying ion currents in the heart*

Evidence that thyroid hormone could indeed change action potential waveforms became available from electrical recordings performed in ventricular cells from guinea pig hearts, that showed decreases in action potential length in the course of hours after application of thyroxine, which then gradually recovered over the course of days (181). In line with these observations, prolongations of action potential durations were observed in hypothyroid rat heart cells (182) and guinea pig ventricular myocytes (183). That thyroid hormone directly effects the electrical properties of heart cells, and not just alters sympathetic receptors was shown by Valcavi et al. (184), who demonstated an increase in the intrinsic activity of the sinus node in hyperthyroid patients that persisted after chemical blockage of autonomous innervation. Patch clamp recordings revealed that in heart cells from neonatal rats (185, 186) and in cat atrial myocytes (187), acute applications of 5-20 nM T3 increased voltage activated sodium currents. Single channel recordings revealed that the application of 5-50 nM T3 induced bursting of Na+ channels in rabbit ventricular myocytes (188). Later studies showed, that T3 increases the sodium channel open probability by binding directly inside the membrane and that the interaction with a pertussis toxin sensitive G-protein greatly enhances this effect (189). More recent experiments by Schmidt et al. (190), confirmed rapid effects of T3 on human hearts, however, suggesting a contribution of the sympathetic nervous system. After a period of prolonged hyperthyroidism in rats, in contrast to acute effects, no changes in Na+current density as well as of inward potassium currents were found. At that time increased rates of rise of the action potentials could be rather explained by an increase in Ca2+-currents and a shortened action potential duration by an increase in a delayed rectifier current (191). Although it is presently not completely understood, which channel regulations exactly determine short and long term effects of thyroid hormone, it is safe to conclude, that an upregulation of voltage activated Na+-, Ca2+ and K+-currents plays a pivotal role in decreases in action potential duration, the acceleration of the heart beat and modulation of contraction amplitude by thyroid hormone.

#### *Influences of thyroid hormone on action potentials and underlying ion currents in the central nervous system*

The influence of thyroid hormone on the electrical properties of neurons has been studied in less detail. The first experiments using whole cell patch clamp recordings were carried out on cultured postnatal rat hippocampal neurons and showed an upregulation of voltagegated Na+-currents (INav) by T3 (192). An upregulation of the density of voltage-gated Na+ currents was also found in acutely isolated neurons from the occipital cortex of hyperthyroid rats and a down regulation observed in cells from hypothyroid rats. The changes in Na+current density led to increased action potential upstroke velocities as well as to enhanced discharge rates in thyroid hormone treated cells in response to identical stimulus strengths (193). Similarly, increases in voltage-gated Na+ currents were observed in human neuroepithelial cells as well as mesenchymal stem cells after incubation with 1 nM T3 for 72h to 6 days in culture. Neuroepithelial cells additionally responded with increases in Ca2+ currents to prolonged T3-treatment (194). In the in *vivo* situation thyroid hormone effects seem to be more complex: Thus in CA1 neurons of the rat hippocampus changes in the bursting pattern have been observed, which could be explained by an upregulation of a low-threshold Ca2+ current (195). Furthermore, consistent with Hoffmann and Dietzel, 2004, decreases in action potential depolarization rate and decreases in discharge rate were observed by thyroid hormone withdrawal. In contrast to the action of thyroid hormone in the heart these authors additionally observed a shortening of action potental duration upon thyroid hormone withdrawal, that could be explained by the upregulation of an A-type potassium current (196). Finally in a somewhat distant animal, in Rohon-Beard neurons from the embryonic zebrafish rapid increases of voltage-gated Na+currents by thyroxine were found (197). This Na+current regulation was shown to be essential for the further development of the embryo and depended on αVβ3 integrin activation and the MAPK (p38) pathway (198). An increase in voltage-gated Na+current density by thyroid hormone would cause a general speeding of mental functions as illustrated in figure 5:

104 Thyroid Hormone

*nervous system* 

sodium current density could as well explain the decreases in peripheral conduction velocities and increases in latencies of evoked potentials found in hypothyroidism (16–24) and reversely the increased amplitudes in hyperthyroidism (177, 178). Since there seems to be an optimal density of sodium channels that ensures maximal neuronal conduction velocity (179), beyond which no further increase or even a slowing of conduction velocity occurs, an upregulation of sodium currents by thyroid hormone could also explain the inconsistent findings concerning latencies of evoked visual potentials in hyperthyroidism, where some authors found decreases in latencies (180) or even increases with increases in thyroid hormone (21, 71, 177, 178). Because of the temperature sensitivity of the activation of sodium and calcium currents the fall in core temperature during hypothyroidism and its

increase in hyperthyroidism could further exacerbate the symptoms (19).

*Influences of thyroid hormone on action potentials and underlying ion currents in the heart* 

Evidence that thyroid hormone could indeed change action potential waveforms became available from electrical recordings performed in ventricular cells from guinea pig hearts, that showed decreases in action potential length in the course of hours after application of thyroxine, which then gradually recovered over the course of days (181). In line with these observations, prolongations of action potential durations were observed in hypothyroid rat heart cells (182) and guinea pig ventricular myocytes (183). That thyroid hormone directly effects the electrical properties of heart cells, and not just alters sympathetic receptors was shown by Valcavi et al. (184), who demonstated an increase in the intrinsic activity of the sinus node in hyperthyroid patients that persisted after chemical blockage of autonomous innervation. Patch clamp recordings revealed that in heart cells from neonatal rats (185, 186) and in cat atrial myocytes (187), acute applications of 5-20 nM T3 increased voltage activated sodium currents. Single channel recordings revealed that the application of 5-50 nM T3 induced bursting of Na+ channels in rabbit ventricular myocytes (188). Later studies showed, that T3 increases the sodium channel open probability by binding directly inside the membrane and that the interaction with a pertussis toxin sensitive G-protein greatly enhances this effect (189). More recent experiments by Schmidt et al. (190), confirmed rapid effects of T3 on human hearts, however, suggesting a contribution of the sympathetic nervous system. After a period of prolonged hyperthyroidism in rats, in contrast to acute effects, no changes in Na+current density as well as of inward potassium currents were found. At that time increased rates of rise of the action potentials could be rather explained by an increase in Ca2+-currents and a shortened action potential duration by an increase in a delayed rectifier current (191). Although it is presently not completely understood, which channel regulations exactly determine short and long term effects of thyroid hormone, it is safe to conclude, that an upregulation of voltage activated Na+-, Ca2+ and K+-currents plays a pivotal role in decreases in action potential duration, the acceleration of the heart beat and modulation of contraction amplitude by thyroid hormone. *Influences of thyroid hormone on action potentials and underlying ion currents in the central* 

The influence of thyroid hormone on the electrical properties of neurons has been studied in less detail. The first experiments using whole cell patch clamp recordings were carried out on cultured postnatal rat hippocampal neurons and showed an upregulation of voltage-

**Figure 5.** Simulation of action potential spread in a hippocampal model neuron, for action potential with high (A-red) and low (A-blue) Na+-current density. A an increase in Na+current density increases action potential depolarization (Aa), amplitude and discharge frequency (Ab). B. Simulation of action potential spread in a ramified neuron 1 ms after application of a stimulus at t=1ms.

B shows the spread for the neuron with high current density and B for the neuron with low current density. The magnified inserts (Ba and Cb) clearly show, that the overshoot of the action potential (light colours) reaches the synapses much earlier in the cell with high current density than in the cell with lower current density (Cb). Simulations performed using the Hodgkin-Huxley equations and the program Neuron version 3.2.3 (199).

Thyroid Hormone Effects on Sensory Perception, Mental Speed, Neuronal Excitability and Ion Channel Regulation 107

**Figure 6.** Illustration of a potential mechanism leading to Na+-current regulation in the hippocampus of rats: T3 stimulates glial cells (stained red by antibodies against GFAP) to secrete growth factors (such as FGF-2) which in turn up-regulate Na+currents in neurons. (stained green by antibodies against βIII tubulin). Thus in addition to regulating the neuronal environment and stimulating synapse formation

Since thyroid hormone has long been known to increase energy expenditure, and about 40% of energy at rest is consumed by activity of the Na+/K+-ATPase (36, 37, 213) many researchers focused on studying effects of thyroid hormone on Na+/K+-ATPase activity and expression. The Na+/K+-ATPase is a heterodimeric membrane spanning protein complex composed of three catalytic alpha subunits (α1, α2 and α3) with molecular weights of ~97- 116kDa and two glycosylated β subunits (β1 and β2 of ~35-55kDa). While the α subunits contain the Na+, K+ and the intracellular ATP binding site, the β subunits are required to insert the catalytic α subunits into the appropriate locations of the cell membrane (214). An intracellular Na+ load of the cell leads to binding of three Na-ions to their intracellular binding sites, thus triggering phosphorylation of the α subunit and inducing a conformational change of the pump to expose the Na-ions to the extracellular surface at the expense of ATP (see e.g. (37)). Thus an increased intracellular Na+-load, as induced by a larger or longer Na+ influx will increase energy consumption by stimulating the demand for ATP. Interestingly, the Na+/K+-ATPase shows a 10-12 fold increase in expression during postnatal development of the brain (215) which parallels the postnatal increase in Na+

The different isoforms of the Na+/K+-ATPase were reported to be distributed in a cell and tissue dependent manner. Thus in brain tissue the α3 isoform transcript is expressed abundantly in comparison with the mRNA for the α1 and α 2 subunits. The α3 expression

glial cells could also be involved in modulating neuronal excitability.

*Regulation of Na+/K+-ATPase expression by thyroid hormone* 

current density (216).

Hypothyreosis also affects a prominent EEG pattern, namely the alpha rhythm (11–15). This may be also related to altered Na+ channel function, since TTX-insensitive Na+ currents of cortical bursting neurones have been implicated in the generation of the alpha rhythm (200). Subsequently, the presence of SCN5A mRNA, encoding the TTX-resistant Na+ channel had been demonstrated in the mammalian brain (201, 202).

It is noteworthy that the mental symptoms observed with the psychophysical tests used in the present study developed gradually, as most prominently demonstrated in the continuous observations on a single person and only recovered with a similar slow time course in the first weeks of hormone resubstitution (see Fig. 1D and 2A). Prolonged recovery phases for the reversal of several of the symptoms accompanying hypothyroidism have been described (20, 84, 203–205) and subjective improvements of well-being, quantified with a "Quality of life-Thyroid scale" were found only after four weeks compared to one week of thyroid hormone replacement (38).

This corresponds to the observation of a lack of acute effects of T3 in hippocampal slices (206) and the observation of a slow upregulation of Na+current density in hippocampal cultures (207). In the later study the Na+current regulation was shown to depend on the presence of glial cells in the culture medium. Thyroid hormone has been shown to induce protein secretion from glial cells (208), including *basic fibroblast growth factor* (FGF-2) (209) and *epidermal growth factor* (210). Furthermore, thyroid hormone has been shown to elevate *nerve growth factor*, *neurotrophin-3* and *brain derived neurotrophic factor* (BDNF) in the brain (see e.g.(211)). A first indication that intermediate steps, including growth factors could be involved in the regulation of Na+currents by thyroid hormone were experiments, that showed, that the effect of T3 on Na+ currents could be reduced by a simultaneous incubation of cultures with antibodies against FGF-2, leading to the hypothesis depicted in Figure 6.

Concerning the action of thyroid hormone on neuronal excitability there seems to be a common finding that the density of voltage-gated Na+currents is up-regulated by thyroid hormone rendering the cells more excitable. This mechanism would explain many of the symptoms observed in thyroid disease, such as slowed peripheral conduction velocity and decreased excitability of the hypothyroid brain. In other tissues, such as various epithelial cells, thyroid hormone could, likewise, play an essential role in the regulation of the expression of amiloride sensitive, epithelial Na+channels (see e.g. (212)). The molecular mechanisms, leading to Na+current upregulation, may however, differ in different species and tissues and warrant further elucidation.

**Figure 6.** Illustration of a potential mechanism leading to Na+-current regulation in the hippocampus of rats: T3 stimulates glial cells (stained red by antibodies against GFAP) to secrete growth factors (such as FGF-2) which in turn up-regulate Na+currents in neurons. (stained green by antibodies against βIII tubulin). Thus in addition to regulating the neuronal environment and stimulating synapse formation glial cells could also be involved in modulating neuronal excitability.

#### *Regulation of Na+/K+-ATPase expression by thyroid hormone*

106 Thyroid Hormone

(199).

B shows the spread for the neuron with high current density and B for the neuron with low current density. The magnified inserts (Ba and Cb) clearly show, that the overshoot of the action potential (light colours) reaches the synapses much earlier in the cell with high current density than in the cell with lower current density (Cb). Simulations performed using the Hodgkin-Huxley equations and the program Neuron version 3.2.3

Hypothyreosis also affects a prominent EEG pattern, namely the alpha rhythm (11–15). This may be also related to altered Na+ channel function, since TTX-insensitive Na+ currents of cortical bursting neurones have been implicated in the generation of the alpha rhythm (200). Subsequently, the presence of SCN5A mRNA, encoding the TTX-resistant Na+ channel had

It is noteworthy that the mental symptoms observed with the psychophysical tests used in the present study developed gradually, as most prominently demonstrated in the continuous observations on a single person and only recovered with a similar slow time course in the first weeks of hormone resubstitution (see Fig. 1D and 2A). Prolonged recovery phases for the reversal of several of the symptoms accompanying hypothyroidism have been described (20, 84, 203–205) and subjective improvements of well-being, quantified with a "Quality of life-Thyroid scale" were found only after four weeks compared to one week of

This corresponds to the observation of a lack of acute effects of T3 in hippocampal slices (206) and the observation of a slow upregulation of Na+current density in hippocampal cultures (207). In the later study the Na+current regulation was shown to depend on the presence of glial cells in the culture medium. Thyroid hormone has been shown to induce protein secretion from glial cells (208), including *basic fibroblast growth factor* (FGF-2) (209) and *epidermal growth factor* (210). Furthermore, thyroid hormone has been shown to elevate *nerve growth factor*, *neurotrophin-3* and *brain derived neurotrophic factor* (BDNF) in the brain (see e.g.(211)). A first indication that intermediate steps, including growth factors could be involved in the regulation of Na+currents by thyroid hormone were experiments, that showed, that the effect of T3 on Na+ currents could be reduced by a simultaneous incubation of cultures with antibodies against FGF-2, leading to the hypothesis depicted

Concerning the action of thyroid hormone on neuronal excitability there seems to be a common finding that the density of voltage-gated Na+currents is up-regulated by thyroid hormone rendering the cells more excitable. This mechanism would explain many of the symptoms observed in thyroid disease, such as slowed peripheral conduction velocity and decreased excitability of the hypothyroid brain. In other tissues, such as various epithelial cells, thyroid hormone could, likewise, play an essential role in the regulation of the expression of amiloride sensitive, epithelial Na+channels (see e.g. (212)). The molecular mechanisms, leading to Na+current upregulation, may however, differ in different species

been demonstrated in the mammalian brain (201, 202).

thyroid hormone replacement (38).

and tissues and warrant further elucidation.

in Figure 6.

Since thyroid hormone has long been known to increase energy expenditure, and about 40% of energy at rest is consumed by activity of the Na+/K+-ATPase (36, 37, 213) many researchers focused on studying effects of thyroid hormone on Na+/K+-ATPase activity and expression. The Na+/K+-ATPase is a heterodimeric membrane spanning protein complex composed of three catalytic alpha subunits (α1, α2 and α3) with molecular weights of ~97- 116kDa and two glycosylated β subunits (β1 and β2 of ~35-55kDa). While the α subunits contain the Na+, K+ and the intracellular ATP binding site, the β subunits are required to insert the catalytic α subunits into the appropriate locations of the cell membrane (214). An intracellular Na+ load of the cell leads to binding of three Na-ions to their intracellular binding sites, thus triggering phosphorylation of the α subunit and inducing a conformational change of the pump to expose the Na-ions to the extracellular surface at the expense of ATP (see e.g. (37)). Thus an increased intracellular Na+-load, as induced by a larger or longer Na+ influx will increase energy consumption by stimulating the demand for ATP. Interestingly, the Na+/K+-ATPase shows a 10-12 fold increase in expression during postnatal development of the brain (215) which parallels the postnatal increase in Na+ current density (216).

The different isoforms of the Na+/K+-ATPase were reported to be distributed in a cell and tissue dependent manner. Thus in brain tissue the α3 isoform transcript is expressed abundantly in comparison with the mRNA for the α1 and α 2 subunits. The α3 expression

increases 10 fold within the first 7 days after birth and remains at this elevated level until the 55th day of age in the rat. In contrast, the mRNA for the α1, α2 and β isoforms reach their maximal expression levels only after the rats are at 25 days old (215).

Thyroid Hormone Effects on Sensory Perception, Mental Speed, Neuronal Excitability and Ion Channel Regulation 109

Thyroid hormone deficiency leads to a general slowing of many body functions, including a slowing of heart rate, a slowing of intestinal movements as well as of thoughts and movements. As demonstrated here in an exemplary fashion on a small sample of patients the most conspicuous symptom to develop during a short period of severe hypothyroidism is a gradual, quantifiable slowing of speech and of critical flicker fusion frequency. Although several explanations at the cellular and molecular level are feasible an intriguing hypothesis is, that a central aspect of the origin of many of these symptoms might be a regulation of the sodium current density that is a key player of neuronal and cellular excitability. In fact, some effects of thyroid hormone can to some extent be blocked by the sodium channel blocker TTX: Thus the upregulation of the membrane Na+/K+ATPase expression in myotubes (227) and skeletal muscle (228) as well as of soma growth in L-GABAergic neurons (230) by thyroid hormone were all to some extent blockable by TTX, suggesting that some effects of thyroid hormone occur downstream of sodium channel regulation. In future it will be exciting to elucidate the full signal cascade involved in the regulation of the different sodium channel subunits as well as to conclusively sort out the primary and the secondary targets of thyroid hormone action. It will be interesting to study

whether some of these thyroid hormone actions decline in the aging brain.

*Department of Molecular Neurobiochemistry, Ruhr-University Bochum, Bochum, Germany* 

*Department of Molecular Neurobiochemistry, Ruhr-University Bochum, Bochum, Germany International Graduate School of Neuroscience (IGSN), Ruhr-University Bochum, Germany* 

*Department of Molecular Neurobiochemistry, Ruhr-University Bochum, Bochum, Germany International Graduate School of Neuroscience (IGSN), Ruhr-University Bochum, Germany* 

*Department of Molecular Neurobiochemistry, Ruhr-University Bochum, Bochum, Germany* 

*Department of Nuclear Medicine, University Hospital, Würzburg, Germany* 

*Department of Nuclear Medicine, University Hospital, Würzburg, Germany* 

*Diagnostische Gemainschaftspraxis Karlstrasse, Karlsruhe, Germany* 

**6. Conclusions** 

**Author details** 

Irmgard D. Dietzel

Sivaraj Mohanasundaram

Vanessa Niederkinkhaus

*PFM Medical AG, Köln, Germany* 

*JM Technische Entwicklungen, Bochum, Germany* 

The late Jens W. Meyer

Christoph Reiners

Christiana Blasl

Gerd Hoffmann

In general, thyroid hormone was found to up-regulate Na+/K+-ATPase activity and expression in many tissues: For instance, in rat cardiomyocytes, T3 was observed to increase the mRNA pattern of Na+/K+-ATPase α1 and β1 subunits 4fold after 48hrs and the α2 mRNA expression even 7fold after 72hrs of treatment (217). A similar effect of T3 was found in a rat liver cell line. Here, a non transformed continuous cell line derived from adult rat liver treated with T3 showed a 1.3 fold increased activity of Na+/K+-ATPase. More specifically, the mRNA expression of the α1 and β1 isoforms of the Na+/K+-ATPase increased 1.5 and 2.9 fold respectively compared with controls maintained in T3 free (hypothyroid) media (218).

In rat brains thyroid hormone has been shown to up-regulate Na+/K+-ATPase activity and protein expression in synaptosomes only in the first two postnatal weeks (219). In addition Schmitt *et al.,* in 1988 showed that the hypothyroid condition reduces the expression of the mRNA for Na+/K+-ATPase α isoforms in rat brain (220). However, observing thyroid hormone effects in identified brain regions in the adult rat indicated, that hypothyroidism could down-regulate Na+/K+-ATPase activity in specific brain regions, such as the adult hippocampus (221, 222). Further experiments showed that the predominant brain cell specific α3 isoform of the Na+/K+-ATPase decreased in hypothyroid rat brain as well and that the relative sensitivity of the different Na+/K+-ATPase α subunits in brain cells for thyroid hormone is α3>α1>α2 (223). The expression of all Na+/K+-ATPase isoforms and their regulation by T3 was also observed in primary neuronal cell cultures of rat brain at the mRNA and protein level using northern and western blot techniques (224). In contrast to neurons, glia cells express α1, α 2 and β1, 2 not α3. The mRNAs as well as the proteins of the four subunits expressed in glia cells showed an upregulation when the cells were grown with the supplement of T3 for 5 and 10 days respectively (225).

Although a T3-responsive element has been found in the promotor region of the α3 subunit (226) two reports on muscle cells indicate, that the regulation of the Na+/K+-ATPase by thyroid hormone might be at least to some extent secondary to an enhanced sodium influx. Thus Brodie and Sampsom (227) observed that a blockage of Na+-influx by tetrodotoxin to block the voltage-gated Na+currents or by amiloride to block further Na+transport routes both reduced the T3-induced increase of 3[H]-ouabain binding sites, which represent membrane inserted Na+/K+-ATPase in cultured myotubes. These results were confirmed by Harrison and Clausen (228) in skeletal muscle, who showed that an increase in saxitoxin binding (reflecting Na+channel density) preceded an increase in 3[H]-ouabain binding (reflecting membrane inserted Na+/K+-ATPases). These experiments indicate a link between Na+ current regulation and the regulation of the Na+/K+-ATPase by thyroid hormone. This is in agreement with other experiments in chick skeletal muscle that suggested that the activation of voltage-gated Na+channels by veratridine leads to an increased biosynthesis of Na+/K+-ATPase in chick myogenic cultures (229). Whether these findings also apply to neurons, or whether some subunits are regulated directly by thyroid hormone receptors and others are regulated by the sodium load of the cells remains, however, to be clarified.
