**3. Methods**

86 Thyroid Hormone

of speech and of visual perception.

molecular level.

interested to test in a small sample of 6 patients, whether already a transient hypothyroid state, induced by 4 weeks of total thyroid hormone withdrawal, would result in detectable changes in the speed of sensory perception and cognitive functions. For this aim we combined different psychophysical tests shown to be sensitive in previous studies of hypothyroidism with some new examinations. While cognitive tests, such as the trail making test as well a calculation task showed a non-significant tendency toward slowing, a more complex visual- spatial performance test revealed a significant slowing of mental function after four weeks of hypothyroidism. The speed of speech was significantly reduced and a fast Fourier analysis showed a shift to lower frequencies in the hypothyroid test persons. A significant decrease in red-green colour fusion frequency was found, indicating an impaired temporal resolution of visual stimuli. Smelling of two odorants tested, odorant discrimination (Sniffin`sticks) and the hearing thresholds were slightly, but insignificantly impaired in the hypothyroid test persons. The results of these tests indicate that the most prominent and first significant clinical symptom to develop in hypothyroidism is a slowing

Slowing of conduction velocity can be explained by a reduced myelination. A second mechanisms is a decrease in voltage-gated sodium current density, leading to a slowed charging of the membrane capacitor thus resulting in a decreased slope of the action potential upstroke velocity which in turn decreases conduction velocity. Although several investigations support the concept that thyroid hormone affects myelination, recently evidence has accumulated, that thyroid hormone also increases sodium current density in neurons from several species. We will thus discuss reports on the regulation of voltage gated ion currents in neurons and muscle cells later in the chapter, which could offer an explanation for the observed slowing of thoughts and movements at the cellular and

Furthermore, it has been known for a long time, that thyroid hormone regulates energy expenditure (see also Yehuda-Shnaidman et al. in this issue). Pumping of Na+ out of the cells has been accounted for the expense of 40% of energy consumed at rest (36, 37). Thus an increased influx of Na+ due to enhanced voltage-gated Na+-influx will most likely also stimulate Na+/K+-ATPase activity, and could also account at least to some extent for the stimulation of an enhanced expression of Na+/K+-ATPase subunits in the membranes. We thus conclude the chapter by reviewing data on the regulation of Na+/K+-ATPase by thyroid

**2. Clinical symptoms during transient severe hypothyroidism quantified** 

To illustrate some conspicuous effects of thyroid hormone on brain function we studied 6 patients after total thyroidectomy for thyroid carcinoma who had discontinued taking thyroid hormone prior to routine diagnostic 131I- scanning and who thus showed a reproducible degree and duration of hypothyroidism. Symptoms described to occur a few weeks after discontinuation of thyroid hormone therapy are changes in peripheral

hormone in the brain and its potential link to Na+ current regulation.

**by psychophysical investigations in adult human test persons** 

*Test persons*. A test battery was developed to allow a relatively fast examination of several aspects of sensory and cognitive function. To integrate the investigation into the normal clinical examination procedures the whole testing protocol was designed to be completed within 1.5 hours. All tests were carried out on 6 patients after thyroid hormone withdrawal for 26 to 28 days and on 6 healthy volunteers which were age (maximal difference: 3 years) and sex matched (with the exception of one female control person for a male patient). Patients were retested after at least 6 weeks of hormone substitution, after obtaining low TSH values. To elaborate the optimal test parameters some of the tests had been performed in more detail on an additional hypothyroid test person, the data of which are included in the appropriate results sections. In the 7 test persons (age 42-64, 4 female, 3 male) TSH-suppressive thyroid hormone substitution after total thyroidectomy and radioiodine therapy for thyroid carcinoma had been discontinued for 26-28 days for routine diagnostic application of 131I. Thyroid hormone levels measured in hypothyroidism were FT3: < 2.0 pmol/l, FT4: <2.6 pmol/l in 6 patients and FT3: 2.6 pmol/l, FT4: 4.8 pmol/l in the remaining patient, TSH was > 80 mU/l in three patients and 48.7 ± 10.4 mU/l (mean ± SE) in the remaining four patients. After 6 - 10 weeks of hormone substitution these values were: FT3: 6.2 ± 0.5 pmol/l, FT4: 26.0 ± 3.0 pmol/l and TSH: 0.09 ± 0.04 mU/l, n=7 (normal ranges: FT3: 3.4 - 7.6 pmol/l (SPART, Amerlex MAB, Johnson & Johnson); FT4: 11 - 23 pmol/l (SPART, Amerlex MAB, Johnson & Johnson), TSH: 0.3 - 4.0 mU/l (IRMA, Dynotest, Brahms). Results are given as means ± standard error. Statistical analysis was performed using paired Student`s t-test. Informed consent was obtained from all individuals before performing the tests.

*Speed of speech*. To investigate possible changes in the speed of speech we asked the test persons to repeat four times the same word as fast as possible (in this case the word "Apfelmus"). They were asked the repeat the series of four words four times and were encouraged to accelerate their speech as much as possible. The four series of words were stored on magnetic tape with a SONY WTC-D6C stereo cassette recorder and analysed offline using a Digidata 1200A analog-digital converter with "Axoscope" software (Axon Instruments). The time needed to pronounce the four words was then read from a digital storage oscilloscope. In addition a fast Fourier analysis was performed on the record of the second syllable ("mus") selected from the two fastest traces obtained from each test person in the hypothyroid and the euthyroid condition. The section of the record to be analysed was selected with "Axoscope" and then analysed with "Origin 5" software.

#### *Tests of cognitive performance*:

a. *Calculation and Correlation*. To test more complex mental performances patients were first handed a sheet of paper and asked to complete a set of 24 simple calculation tasks of third grade difficulty (like: 23+11=?). Each result of a calculus task was assigned one of five colours (yellow, red, green, dark and light blue). After completing the calculation task the test persons were asked to fill a second form, consisting of an outline drawing containing 54 numbered areas, where each number equalled one of the results of the preceding calculus task (some numbers were used several times). The patients were handed coloured pencils and asked to assign the appropriate colour from the result of the calculus task to each of the numbers given in the drawing. This procedure finally resulted in the appearance of a meaningful picture (in this case a boat). The time taken by the patients to complete the calculus task and to assign the colours to the figures in the drawing was monitored.

Thyroid Hormone Effects on Sensory Perception,

Mental Speed, Neuronal Excitability and Ion Channel Regulation 89

per minute was electronically counted. Increasing the speed of rotation first gave the impression of a luminance flicker. A further increase in the speed of rotation resulted in the impression of a homogenous dark yellow colour. The patients were asked to give a sign at the frequency were they saw the first signs of a luminance flicker and as soon as they perceived the impression of a homogenous yellow colour. Each test was repeated three times starting with low frequencies and the average value of the three

*Sense of Smell*. Two tests were used to detect possible changes in the sense of smell: First a test for the recognition and discrimination of 16 different odorants, including familial smells like cinnamon and rather unusual flavours, like leather, was used. Then the threshold of perception was tested using two different odours, one that tests the excitation of the olfactory nerve (phenylethyl alcohol (Phe)), smelling like rose, and one, exciting both, trigeminal and olfactory nerves (eugenol (Eu)), Sniffing´sticks, (45)). In brief, for the odorant tests, caps were removed from plastic sticks containing odorants of different concentrations filled into the sticks ending in felt tips extruding about 5 mm from the tip of the stick. A stick was gently moved at a distance of about 1 cm below the nostril of the test person. Sticks containing the test solution in ascending concentration (descending numbers on the stick) were presented to the test person in series of three sticks, two of which contained distilled water. The threshold was defined as the concentration at which the patient

*Hearing threshold*. Hearing thresholds were determined for 8 different frequencies using an Ascom Audiosys Maico ST20 audiometer. Changes in threshold for sinusoidal tones of 1

*Speed of speech.* Figures 1A and B show digitized traces of speech records of a female test person in hypothyroidism (upper trace, a) and after hormone substitution (lower trace, b). After hormone substitution this test person pronounced the four words faster. As shown in Fig. 1C, on average the test persons needed a significantly longer time to pronounce the same words in the hypothyroid condition as compared to the euthyroid control persons or after thyroid hormone substitution. Figure 1D gives a more elaborate example of the development of the slowing of speech during hormone withdrawal and resubstitution. Here an additional test person was asked to repeat a short poem in regular intervals at maximal speed and the time taken to complete this poem was recorded. During hormone withdrawal the time needed to finish the poem became increasing longer. During resubstitution with thyroid hormone the time to finish the poem gradually decreased during the following month. To find out whether the increase in speed of speech during hormone resubstitution was accompanied by an increase in pitch a fast Fourier analysis was performed on the syllable "mus" (encircled by the rectangles in Fig. 1A). The analysis of the pronounciation of this syllable, consisting with predominant amplitude of the noun "u" showed several clear frequency peaks (Fig. 1B). The records from four of the five male subjects included in the

correctly recognised the odour in two out of three presentations.

determinations was noted.

and 8 kHz were evaluated.

**3.1. Experimental results** 

b. *Trail making*. The test consisted of a piece of DIN A4 paper, containing randomly distributed numbers (24 pt size, black, surrounded by a black circle). The paper was placed on a table in front of the test persons who were asked to connect the numbers from one to 25 (version A). In version B numbers from 1 to 13 and letters from A to L were distributed randomly and the test persons asked to connect them alternating between the numbers and the succession of the alphabet, e.g., 1-A 2-B- 3-C etc. The time needed for completion of the test was recorded (44).

*Tests to determine time resolution of visual perception and colour contrast perception:* 


per minute was electronically counted. Increasing the speed of rotation first gave the impression of a luminance flicker. A further increase in the speed of rotation resulted in the impression of a homogenous dark yellow colour. The patients were asked to give a sign at the frequency were they saw the first signs of a luminance flicker and as soon as they perceived the impression of a homogenous yellow colour. Each test was repeated three times starting with low frequencies and the average value of the three determinations was noted.

*Sense of Smell*. Two tests were used to detect possible changes in the sense of smell: First a test for the recognition and discrimination of 16 different odorants, including familial smells like cinnamon and rather unusual flavours, like leather, was used. Then the threshold of perception was tested using two different odours, one that tests the excitation of the olfactory nerve (phenylethyl alcohol (Phe)), smelling like rose, and one, exciting both, trigeminal and olfactory nerves (eugenol (Eu)), Sniffing´sticks, (45)). In brief, for the odorant tests, caps were removed from plastic sticks containing odorants of different concentrations filled into the sticks ending in felt tips extruding about 5 mm from the tip of the stick. A stick was gently moved at a distance of about 1 cm below the nostril of the test person. Sticks containing the test solution in ascending concentration (descending numbers on the stick) were presented to the test person in series of three sticks, two of which contained distilled water. The threshold was defined as the concentration at which the patient correctly recognised the odour in two out of three presentations.

*Hearing threshold*. Hearing thresholds were determined for 8 different frequencies using an Ascom Audiosys Maico ST20 audiometer. Changes in threshold for sinusoidal tones of 1 and 8 kHz were evaluated.
