Thyroid hormone regulates excitability in central neurons from postnatal rats

Neuroscience 125 (2004) 369 –379

THYROID HORMONE REGULATES NEURONS FROM POSTNATAL RATS G. HOFFMANN1 AND I. D. DIETZEL*

EXCITABILITY

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CENTRAL

creased frequencies of EEG-waves (Schlutter et al., 1978). Most studies carried out so far focused predominantly on effects of thyroid hormone on changes in the cytoarchitecture and the expression of proteins involved in transmitter metabolism, synapse formation and myelination (Thompson and Potter, 2000; Bernal, 2002; Ko¨nig and Moura Neto, 2002; Zoeller et al., 2002). However, there has been no conclusive explanation for the biophysical mechanisms underlying the conspicuous slowing of several EEGparameters and mental function in hypothyroidism. Neuronal excitability is determined in the first place by the Na⫹-current density. We have previously obtained evidence that thyroid hormone increases the Na⫹-current density in cultured hippocampal neurons from postnatal rats leaving delayed rectifier and transient K⫹ currents unchanged (Potthoff and Dietzel, 1997). It had, however, remained unresolved, whether Na⫹ currents can be regulated by thyroid hormone to an extent large enough to modify the shapes of action potentials and neuronal firing frequencies. In addition, it had not been tested, whether the increase of the sodium current density by thyroid hormone occurs at all holding potentials or is rather caused by a shift of the voltage dependence of inactivation to more depolarized values. It had also not been investigated, whether thyroid hormone regulates voltage-activated sodium currents only in cells from hippocampus or also in cortical neurons. A further open question was, whether the regulation of Na⫹ currents observed in culture also occurs in vivo.

Department of Molecular Neurobiochemistry, Ruhr-University Bochum, NC7-170, Universita¨tsstrasse 150, D-44780 Bochum, Germany

Abstract—A lack of thyroid hormone in the postnatal period causes an irreversible mental retardation, characterized by a slowing of thoughts and movements accompanied by prolonged latencies of several evoked potentials and slowed electroencephalographic rhythms. Here we show that in cultured hippocampal and cortical neurons from postnatal rats treatment with thyroid hormone not only up-regulates Naⴙ-current densities but also increases rates of rise, amplitudes and firing frequencies of action potentials. Furthermore, we show that the regulation of the Naⴙ-current density by thyroid hormones also occurs in vivo: recordings from acutely isolated cortical neurons obtained from hypothyroid, euthyroid and hyperthyroid postnatal rats showed that hypothyroidism decreases the ratio of Naⴙ inward- to Kⴙ outward-currents while hyperthyroidism upregulates Naⴙ-currents with respect to Kⴙ-currents. Our observation of a regulation of neuronal excitability by thyroid hormone offers a direct explanation for the origin of various neurological symptoms related to thyroid dysfunction. © 2004 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: hippocampus, cortex, triiodothyronine, sodium current regulation, action potentials, firing frequency.

Inadequate supply of the developing brain with thyroid hormone leads to irreversible mental retardation. An especially prominent symptom caused by thyroid hormone deficiency is a general mental slowing that is accompanied by increased latencies of several types of evoked potentials (Bradley et al., 1961; Laureau et al., 1987; NorcrossNechay et al., 1989; Albee et al., 1989), decreases in electroencephalogram (EEG) amplitudes and frequencies (Bertrand et al., 1938; Bradley et al., 1960; Lansing and Trunnell, 1963) as well as a slowing of conduction velocities of peripheral nerves (De Vries et al., 1986; Beghi et al., 1989) in rats as well as humans. An excess of thyroid hormone on the other hand leads to nervousness, restlessness and tremor (Kudrjavcev, 1978) accompanied by in-

EXPERIMENTAL PROCEDURES Preparation of cultures Hippocampi and cortices were obtained from 3 to 5-day-old rat pups under sterile conditions, collected in ice-cold modified phosphate buffered saline, containing (in mM): NaCl 137, KCl 0.2, Na2HPO4 10.1, KH2PO4 1.8, HEPES 10, pyruvate 1, glucose 10, L-alanyl-glutamine 1, desoxyribonuclease I 1 ␮g/ml, bovine serum albumin 1 mg/ml, Phenole Red 2.5 mg/l. After addition of 30 ␮l 2.5% trypsin tissue was incubated under gentle agitation for 30 min at 37 °C. Cells were triturated through plastic pipette tips, the dissociated cells collected in 8 ml Dulbecco’s minimal essential medium centrifuged at 200 g at 4 °C for 10 min and the pellet resuspended in B18 medium (Brewer and Cotman, 1989). Cells were seeded on 3.5 cm plastic Petri dishes at a density of 6 –9⫻105 cells/cm2 in B18 medium supplemented with 10 ␮l of a penicillin (104 IU/ml)–streptomycin (104 ␮g/ml) stock solution. Cells were stored in a B5060 incubator (Heraeus, Hanau, Germany) at 37 °C and 5% CO2 in humidified air. 3,3⬘,5-Triiodo-Lthyronine (T3) was dissolved in a 1:2 ethanol–1 N HCl mixture to a concentration of 0.2 mg/ml. A stock solution of 1 ␮l of this solution/ml B18 was prepared yielding a concentration of approximately 300 nM T3. Beginning with day 4 in culture 10% of this

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Present address: Cytocentrics CCS GmbH, D-72770 Reutlingen, Germany. *Corresponding author. Tel: ⫹49-234-32-25803; fax: ⫹49-234-32-14105. E-mail address: [email protected] (I. D. Dietzel). Abbreviations: EEG, electroencephalogram; EGTA, ethylene glycolbis-2-aminoethylether-N,N,N⬘,N⬘-tetraacetic acid; fT3, free 3,3⬘,5-triiodo-L-thyronine-concentration; HEPES, N-2-hydoxyethylpiperazine-N⬘-2-ethanesulfonic acid; NGF, nerve growth factor; PTU, 6-n-propyl-2-thiouracil; T3, 3,3⬘,5-triiodo-L-thyronine.

0306-4522/04$30.00⫹0.00 © 2004 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2004.01.047

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solution was added to the culture dishes while the control cells were incubated with 10% of an analogous stock solution on the basis of the solvent. A preincubation with 30 nM T3, about six times larger than in our previous investigation was chosen to obtain a near maximal effect on Na⫹-current regulation. This concentration was chosen on the basis of previous studies of various authors showing that effects of T3 on cultured cells start with concentrations of about 1 nM (about the physiological total T3-concentration in rats, mice and guinea-pigs) (Felzen et al., 1991; Di Meo et al., 1994; Shimoni et al., 1997; Knipper et al., 1999) and reach maximal values at 10 –100 nM (Honegger and Lenoir, 1980; Brodie and Sampson, 1989; Harris et al., 1991; Filipcˇik et al.,1994; Wickenden et al., 1997). Cells were investigated following 2– 4 days of incubation in T3 containing or T3 free medium.

Induction of hyper- and hypothyroidism To minimize the number of animals treated in vivo most experiments were performed on cultured cells. To verify whether the effects observed by thyroid hormone in culture occur in vivo as well some experiments were performed on acutely isolated cells obtained from hypo- or hyperthyroid rat pups. The procedures for treating the animals were carried out in accordance with the German animal protection law (approval no 23.8720 Nr. 36.6, Bezirksregierung Arnsberg). Rat pups were made hypothyroid by treatment of the mother with 0.05% 6-n-propyl-2-thiouracil (PTU) and 5% glucose in the drinking water beginning with the 18th day of gestation. Rat pups were made hyperthyroid by daily s.c. injections of 2 ␮g T3 and 2 ␮g thyroxine, dissolved in 100 ␮l phosphate buffered saline (see e.g.Wibo et al., 1995; Shimoni et al., 1997). The effect of these treatments on plasma thyroid hormone levels was verified by immunochemical determination of free thyroid hormone (fT3) levels. In pups, the mothers of which had been treated with PTU in the drinking water fT3 amounted to 0.9⫾0.2 pM, in controls it was 2.4⫾0.2 pM and in thyroid hormone-treated pups fT3 was ⬎40 pM (three determinations in plasma collected from animals at p5–p6; SPART, Amerlex MAB*fT3, Trinity Biotech, Wicklow, Ireland). Likewise, the supplementation of culture dishes with 30 nM T3 resulted in fT3 concentrations larger than 40 pM (four determinations in culture dish supernatants) whereas in T3-free culture dishes fT3 was undetectable.

Acute dissociation Cells from hypo-, hyper- and euthyroid rats were dissociated similarly as described by Alzheimer et al. (1993). For each recording session two postnatal rats, aged P2–P5 were decapitated and the cortex removed with fine dissection forceps. Slices (500 ␮m thick) of the occipital cortex were cut using a TSE Vibrating microtome 500-2C2 (TSE, Bad Homburg, Germany). The white matter was removed with fine forceps and the slices transferred to 20 ml ice-cold dissociation solution, containing in mM: NaCl 130, KCl 5, CaCl2 2, MgCl2 1, HEPES 20, glucose 25, pH adjusted to 7.4, saturated with 5% CO2 in O2. Following addition of 500 ␮l trypsin (2.5%) and 80 ␮l papain (391 IU/ml) the temperature of the solution was elevated to 32 °C to increase the enzyme action and the slices were incubated under gentle agitation and saturation with O2 and 5% CO2. After 1.5 h the solution was renewed, the incubation continued for another hour under gentle agitation and terminated by an incubation for a further hour without stirring. Two to three slices were dissociated by gentle trituration through firepolished Pasteur pipettes with decreasing orifices in carbogenesaturated solution and the cell suspension was pipetted into a plastic Petri dish. After half an hour at room temperature in the dissociation solution without enzymes the cells had sufficiently attached to the plastic bottom of the dish to be ready for recording with patch pipettes.

Recording conditions Whole cell recordings were performed with borosilicate pipettes (GB 155- 8P; Science Products, Hofheim, Germany) pulled with a PP-830 puller (Narishige Europe, London, UK) to resistances of approximately 5 M⍀ after filling. Pipette solutions contained in mM: K⫹ gluconate 140, NaCl 5, MgCl2 1.0, CaCl2 0.2, EGTA 2.0, HEPES 15.0, pH 7.4, bath solution: NaCl 145, KCl 4, MgCl2 2, CaCl2 3, glucose 10, HEPES 10, pH 7.4. Reported potentials were corrected for a measured liquid junction potential of bath with respect to pipette solution of 15 mV (see e.g. Barry and Lynch, 1991). Recordings were performed at room temperature using a home-build EPC-5-type patch clamp amplifier without a series resistance compensation circuit. The series resistance causes a shift in the peak of the sodium current in the I/V curve to more hyperpolarized values which increases with the size of the current being measured (see e.g. Marty and Neher, 1995). Assuming a series resistance of 20 M⍀, as was typically recorded from the instantaneous current surge following a voltage jump after establishing the whole cell configuration, and a current of 300 pA in thyroid hormone-treated cells the resulting steady-state voltage error was 6 mV, which is within the experimental error. In control cells showing currents of less than 100 pA it amounted to less then 2 mV. Since the series resistance error shifts the peak Na⫹current to more negative voltages, including cells with larger series resistance errors would lead to an underestimation of the Na⫹-current measured at a voltage of ⫺20 mV in the T3-treated cells, thus leading to an underestimation of the T3 effects on the Na⫹-current densities. Intracellular recordings were performed using an intracellular recording amplifier with capacitance compensation and current pump unit. To obtain an optimal measurement of the action potential rise time the head stage had been equipped with a low noise high input impedance (1013 ⍀), high speed amplifier (BBOPA 621) and a fast low noise main stage amplifier (kindly build by Jens Meyer for this project). Recordings were performed using patch electrodes identical to those used for the whole cell recordings. The use of the intracellular recording amplifier did not allow us to judge the quality of the seal properly. Hence all recordings of cells with a resting potential positive to ⫺50 mV were discarded. To minimize an influence of variations of the resting membrane potential on the action potential shapes all cells were maintained near ⫺70 mV by current injection. Nevertheless, in cells with poorer seals, the short circuit of the stimulus current could have led to an actually smaller stimulus current due to the larger leaks causing a slower action potential and frequency response in this cell and could hence have increased the scatter of the data. In addition a large source of scatter could have arisen due to the difficulty of identifying cell types by morphological criteria in the cultures. Most of our recordings were performed on cells with an ellipsoid soma and two primary dendrites. Although GABAergic interneurons have been shown to grow out fewer primary neurites on polylysine-coated surfaces than pyramidal-type cells (Stichel and Mu¨ller, 1992), they comprise only about 10% (Stichel and Mu¨ller, 1992) to 20% of the neuronal population in neocortical cultures (Huettner and Baughman, 1986). In hippocampal cultures about 6% are GABAergic and assume a characteristic triangular soma shape and fewer primary processes with longer dendritic segments after 3 weeks in culture (Benson et al., 1994). Although our preferential choice of bipolar cells could have increased the number of GABAergic neurons recorded from to more than 20%, an unambiguous identification of the cells recorded from was not possible at that early stage. Since most of the non-GABAergic neurons show only two primary neurites at an early stage in culture as well (Stichel and Mu¨ller, 1992) we assume that we recorded from a heterogeneous population of cells the majority of which represents pyramidal cells.

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Fig. 1. Influence of thyroid hormone on whole cell currents. (A) Current traces evoked by step depolarizations (arrows) to test potentials of ⫺80 to ⫹25 mV, holding potential: ⫺85 mV. Cell cultured in 0 mM T3. (B) Recordings from cell in sister culture supplemented for 3 days with 30 nM T3. (C) Average peak inward current versus test potential for recordings from 10 cells cultured in 0 mM T3 (white squares) and 10 cells cultured in 30 nM T3-(black squares). (A–C) From hippocampal cells. (D) Average of the ratios of peak inward currents (measured at test potentials of ⫺20 mV) to outward currents (measured after 60 ms of depolarization at test potentials of ⫹25 mV). (E) Na⫹-current densities (measured at test potentials of ⫺20 mV normalized to membrane capacity). Numbers in bars in (D) indicate numbers of cells recorded from; numbers in (E) indicate numbers of preparations. To calculate the values shown in (E) current densities obtained for several successful recordings from each preparation were averaged first and then averaged over the different preparations to reduce the scatter caused by recording from varying numbers of cells in different recording sessions. Error bars indicate S.E.M. (* P⬍0.05, ** P⬍0.01).

Data were digitized with a Digidata 1200 board and PClamp 6 Software (Axon Instruments, Union City, CA, USA), capacitive artifacts and leak currents subtracted with a P/N protocol and stored on an IBM compatible PC. Further data evaluation was performed with Origin 5.0 software. Peak Na⫹-currents were measured at a potential of ⫺20 mV and K⫹ currents were measured at a potential of ⫹25 mV after 60 ms of depolarisation. Statistical comparisons used Student’s t-test and errors are given as ⫾S.E.M.

RESULTS Cultured hippocampal and cortical cells were investigated using the patch clamp technique in the whole cell configuration following 2– 4 days of incubation in B18 medium containing 0 nM or 30 nM T3. In accordance with our preceding observations (Potthoff and Dietzel, 1997) pretreatment with T3 increased the amplitude of voltage-gated Na⫹-currents (Fig. 1A,B). As shown in Fig. 1B, Na⫹ currents activated at potentials positive to ⫺55 mV and reached peak values at about ⫺20 mV. The approximately 5 mV steeper slope of activation of the averaged Na⫹ current in the T3-treated cells corresponds to the expected distortion of the of the current voltage relationship due to the series resistance of ⱕ20 M⍀ during recording. Hence no obvious differences in the voltage-dependence of activation were noticed. Because our previous investigations

had shown that K⫹-currents are unaffected by T3 in postnatal hippocampal neurons we did not separate Na⫹- and K⫹-currents and investigated the ratio of the Na⫹ to the K⫹-currents (Fig. 1D). By normalizing the Na⫹ to the K⫹current density this procedure reduces the large scatter between the different cells in a dish. The ratio of the peak Na⫹ to the persistent K⫹ current was 0.32⫾0.01 (n⫽21) in hippocampal control cells and 1.45⫾0.06 (n⫽30) after T3treatment (eight different preparations; P⬍10⫺6; Fig. 1D). Normalized to membrane capacity the Na⫹-current density increased from 1.43⫾0.50 pA/pF (n⫽8) to 4.34⫾0.86 pA/pF (averages of n⫽8 preparations; P⬍0.05; Fig. 1E). Hence T3 treatment increases the Na⫹/K⫹-current ratio by a factor of 4.5 and the Na⫹-current density on the average by a factor of 3.0 in hippocampal cells. We now tested, whether the regulation of Na⫹-currents also occurs in cultured cells from rat neocortex. In cortical neurons the ratio of the Na⫹-current with respect to the K⫹-current amounted to 0.19⫾0.01 (n⫽24) in untreated cells compared with 0.46⫾0.03 (n⫽12) after T3-treatment (seven different preparations; P⬍10⫺5; Fig. 1D). The Na⫹-current density increased from 0.91⫾0.32 pA/pF (n⫽7) to 1.31⫾0.31 pA/pF (averages of n⫽7 preparations; P⬎0.05; Fig. 1E). Hence the ratio of the Na⫹ to the K⫹-currents increased by a factor of 2.4. Due to the larger scatter in the

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Fig. 2. Influence of thyroid hormone on steady-state inactivation of Na⫹-currents. (A) Pulse protocol: holding potential: ⫺85 mV, incrementing prepulses in steps of 5 mV applied for 200 ms starting with hyperpolarizations to ⫺105 mV followed by a depolarizing test pulse to ⫺20 mV applied for 50 ms. Recordings from hippocampal (B) and cortical (C) neurons. Families of inward current recordings from control cells (Ba, Ca) and T3-treated cells (Bb, Cb). (Bc, Cc) Peak inward currents normalized to maximal current versus prepulse potential (32 T3-treated cells [black squares] and 21 control cells [white squares] averaged in hippocampal cultures and 12 T3-incubated neurons [black squares] and 24 control cells [white squares]) investigated in cortical cultures. Solid lines fitted to a modified Boltzmann equation with a least squares algorithm. Note, that at a holding potential of ⫺85 mV (stippled line) the shift in the steady state inactivation curve causes at most a change in the amplitude of the inward current by 3.7%.

Na⫹-current density recording the increase by a factor of 1.4 in cortical neurons did not reach statistical significance. An increase in the Na⫹-current by thyroid hormone incubation could, in principle, be caused by a shift of the voltage dependence of inactivation of the Na⫹-current to more depolarized values, thus allowing the activation of more Na⫹ channels from the same holding potential in T3-treated cells. Shifts in the voltage dependence of inactivation to more depolarizing values have indeed been observed after status epilepticus (Ellerkmann et al., 2003), and are induced in hyperpolarizing direction by the protein kinase C activators phorbol-12,13-diacetate (Chizhmakov and Klee, 1994) and 1-oleoyl-2-acetyl-sn-glycerol (Franceschetti et al., 2000). We thus investigated the steady state inactivation of Na⫹-currents in control cells and in T3preincubated cells using the prepulse protocol shown in Fig. 2A. The preincubation with T3 shifted the point of half maximal inactivation by an average of 3 mV to depolarized values in hippocampal neurons, causing no change in the amplitude of the inward current at a holding potential of ⫺85 mV (Fig. 2Bc). In cortical cells a shift of the steady state inactivation curve by 5 mV to the right was observed, explaining at most an increase in the relative Na⫹-current by 3.7% and not by at least a factor of 40%, as presently observed (Fig. 2Cc). Thyroid hormone preincubation thus did not change the percentage of the Na⫹-current evoked from a holding potential of ⫺85 mV.

The position of the steady-state inactivation curve with a voltage of half inactivation of about ⫺53 mV corresponded to that reported in hippocampal progenitor cells (Sah et al., 1997) and is in line with other reports suggesting that in immature hippocampal neurons the steady-state inactivation curve is about 10 mV more depolarized than in mature cells which show voltages of half inactivation of about ⫺66 mV (Cummins et al., 1994; Costa, 1996). We now tested whether thyroid hormone influences neuronal excitability performing intracellular action potential recordings with patch pipettes in the whole cell configuration. To increase the yield of spiking neurons cultures were prepared from slightly older, 4 – 6 day old rats, precultured for 4 days in B18 and investigated after 2– 4 days of incubation in the presence or absence of 30 nM T3. Spikes of cells pretreated with T3 showed significantly faster rates of rise (Fig. 3Aa, Ba). The rise time from 10% to 90% of the action potential amplitude amounted to 1.35⫾0.09 ms in T3-treated hippocampal cells (n⫽45) and to 1.63⫾0.10 ms in control cells (n⫽44; P⬍0.05; Fig. 3Ab). In cortical cells the rise time after T3-treatment was 1.71⫾0.13 ms (n⫽37) compared with 3.41⫾0.66 ms in untreated cells (n⫽33; P⬍0.05; Fig. 3Bb). The amplitudes of the action potentials (measured as voltage difference between peak voltage and maximum of the after-hyperpolarization) were significantly larger in T3treated neurons. Action potential amplitudes amounted to

G. Hoffmann and I. D. Dietzel / Neuroscience 125 (2004) 369 –379

Fig. 3. Influence of thyroid hormone on rise times and amplitudes of action potentials. Shapes of action potentials from hippocampal (Aa) and cortical cells (Ba) cultured in the presence or absence of 30 nM T3. Stimulation current: 200 pA, holding potential: ⫺70 mV. Averages of rise times (10 –90%) of action potentials of hippocampal (Ab) and cortical cells (Bb). Averages of action potential amplitudes in hippocampal (Ac) and cortical cells (Bc). Numbers in the bar charts indicate the numbers of cells investigated. Error bars represent S.E.M. (* P⬍0.05, ** P⬍0.01).

50.8⫾1.7 mV (n⫽45) in T3-treated hippocampal cells and to 46.4⫾0.9 mV (n⫽44) in controls (P⬍0.002; Fig. 3Ac). In cortical cells action potential amplitudes were 45.3⫾2.8 mV (n⫽37) after T3 treatment compared with 37.1⫾3.7 mV (n⫽33) in control cells (P⬍0.05; Fig. 3Bc). The effect of thyroid hormone on neuronal discharge rates was investigated by applying stimulus currents of increasing amplitudes and monitoring the frequency response of the neurons. As shown in Fig. 4Ad and Bd the effect of thyroid hormone on firing frequency increased with increasing stimulus strength. Whereas the firing frequencies were similar in control and T3-treated cells using small stimulus currents maximal firing frequencies elicited with larger stimulus currents were larger in T3-treated neurons. In the cells where a complete frequency versus stimulation current curve could be recorded the minimal interval between successive action potentials at maximal firing frequency significantly decreased from 39.8⫾1.5 ms (n⫽17) in hippocampal control cells to 32.1⫾0.9 ms (n⫽16) in T3-treated cells (P⬍0.005; Fig. 4Ac). In cortical cells it decreased from 30.5⫾1.2 ms (n⫽13) to 24.1⫾0.9 ms (n⫽11; P⬍0.005; Fig. 4Bc). Finally we tested whether thyroid hormone regulates Na⫹-currents in vivo as well. We treated rat pups either with the thyreostatic drug PTU or with thyroid hormone and

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measured the ion currents in acutely dissociated cells from the occipital cortex in the whole cell configuration (Fig. 5). Cells from PTU-treated hypothyroid rats displayed significantly smaller membrane capacitances of 25.1⫾1.2 pF (n⫽26) compared with those of cells obtained from control rats (31.5⫾1.2 pF; n⫽21; P⬍0.002; Fig. 5Bc). The Na⫹current densities were 6.2⫾1.2 pA/pF (n⫽26) in cells from PTU-treated animals and 9.7⫾1.5 pA/pF (n⫽21) in cells from control animals (P⫽0.08; Fig. 5Bb). The Na⫹/K⫹current ratios were significantly smaller (0.6⫾0.1, n⫽26) in cells from PTU-treated animals than in cells from control animals (0.9⫾0.1; n⫽21; P⬍0.001; Fig. 5Ba). To test whether a surplus of thyroid hormone on the other hand increases Na⫹-current densities newborn rats received daily s.c. injections of 2 ␮g T3 and 2 ␮g T4. This treatment resulted in a significant increase of the Na⫹-current density from 9.7⫾1.5 pA/pF (n⫽21) to 14.5⫾1.6 pA/pF (n⫽21) in cells from thyroid hormone-treated rats (P⬍0.04; Fig. 5Bb). Likewise, the Na⫹/K⫹-current ratio increased from 0.9⫾0.1 (n⫽21) to 1.8⫾0.2 (n⫽21) after thyroid hormone treatment (P⬍0.0001; Fig. 5Ba). The capacitance changed from 31.5⫾1.5 pF (n⫽21) to 31.8⫾2.3 pF (n⫽21) after treatment (P⫽0.9; Fig. 5Bc). Compared with cells from control rats T3-treated rats showed significant increases in all three parameters, Na⫹/K⫹-current ratios (P⬍10⫺7), Na⫹-current densities (P⬍10⫺3) as well as membrane capacitances (P⬍0.01) as compared with PTU-treated rat pups. This indicates that in vivo deviations from the physiological level of thyroid hormone allow a regulation of the Na⫹-current density in both directions.

DISCUSSION Various observations have previously indicated an influence of thyroid hormone on neuronal excitability: In addition to effects on electroencephalographic parameters (see introduction) thyroid hormone has been reported to lower seizure threshold in animals as well as humans (Timiras et al., 1955; Swanson et al., 1981; Kahaly et al., 1989; Sandrini et al., 1992; Radetti et al., 1993), decrease the sensitivity to anesthetics in rats (Rutsch, 1933), increase the excitability of peripheral nerves (Horsten and Boeles, 1949; Makii et al., 2002) and increase neuronal discharge rates in cats (Davidoff and Ruskin, 1972). Effects of thyroid hormone on transmitter metabolism and cytoarchitecture Thyroid hormone has been shown to regulate several neurotransmitter systems, including the development of cholinergic terminals and enzymes for cholinergic transmission in rat forebrain, hippocampus and amygdala (Rastogi et al., 1977; Honegger and Lenoir, 1980; Oh et al., 1991). The effect of thyroid hormone on cholinergic transmission could contribute to the known memory impairments observed in hypothyroidism (Smith et al., 2002). Furthermore, the development of the peripheral noradrenergic system is profoundly depressed in hypothyroidism (Slotkin and Slepetis, 1984) and decreased numbers of ␤-adrenergic receptors have been found in brains of de-

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Fig. 4. Influence of thyroid hormone on action potential firing frequencies. Maximal firing frequency of a control neuron after 1 week in culture from hippocampus (Aa) and from cortex (Ba). Maximal firing frequency of a neuron from a sister culture incubated for the last 3 days with 30 nM T3 from hippocampus (Ab) and from cortex (Bb). Average action potential intervals for maximal firing frequency for hippocampal (Ac) and cortical cells (Bc). Numbers of cells evaluated are shown in the bar charts. Frequency versus stimulus current for hippocampal (Ad) and cortical cells (Bd). n⫽numbers as given in (Ac) and (Bc). Error bars indicate S.E.M. (** P⬍0.01).

veloping hypothyroid rats (Smith et al., 1980; Gross et al., 1980). Serotonin levels are decreased by neonatal hypothyroidism (Rastogi and Singhal, 1978) and a downregulation of serotonin release and 5-HT2 receptor sensitivity in cortical neurons has been suggested as cause for the mood disorders accompanying hypothyroidism (Bauer et al., 2002). Finally, a reduction of the number of GABAAreceptors has been described in hyperthyroid adult rats (Sandrini et al., 1992) and T3 has been shown to directly inhibit Cl⫺-currents through GABAA-receptors in high concentrations (10 nM to 5 ␮M; Martin et al., 1996), which could contribute to the increases in excitability observed in hyperthyroidism. Hypothyroidism during gestation causes impairments of neuronal migration and disturbances in the architecture of the cortical layers (see e.g. Lavado-Autric et al., 2003). Postnatal hypothyroidism still causes changes in neuronal morphology, such as a decrease in the soma size as well as a reduced neurite extension, neuritic spines and number of branching points (Ruiz-Marcos et al., 1980; Rami et al., 1986). First morphological changes of hippocampal neurons have been noticed at postnatal day 6, slightly later

than the effects of T3 on Na⫹-currents observed in the present study (Rami et al., 1986). Likewise, no obvious influences on the morphology and survival of cells have been observed in low density hippocampal and cortical cultures within 3 days of incubation with up to 100 nM T3 (Filipe`ik et al., 1994). Although hypothyroidism led to smaller membrane capacitances in the cells acutely dissociated from hypothyroid pups in the present study, we had observed no significant changes in capacitance and soma size in cultured hippocampal cells treated with 5.2 nM of T3 (Potthoff and Dietzel, 1997). Hence at least in culture, the effects of T3 on Na⫹-currents seem to precede measurable morphological changes. Evidence for a contribution of Naⴙ-current regulation to some physiological effects of thyroid hormone Smaller nerve fibers, decreased numbers of releasable vesicles, decreased numbers of receptors as well as decreased myelination could in fact result in a slowing of conduction velocity and reduced evoked potentials observed in hypothyroidism. On the other hand, a down-

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Fig. 5. Influence of thyroid hormone on whole cell currents in acutely dissociated neurons. Voltage-activated currents from a cortical cell obtained from (Aa) hypothyroid pup after treatment of the mother with PTU, (Ab) control rat, (Ac) pup treated by daily s.c. injections of 2 ␮g T3 and 2 ␮g T4, all recordings from 4 day old pups. (Ba) Na⫹/K⫹current ratios, (Bb) Na⫹-current densities, (Bc) membrane capacitances of cells from PTU-treated, control and T3-treated pups. Numbers in bars indicate numbers of cells recorded from. Error bars represent S.E.M. (* P⬍0.05, ** P⬍0.01).

regulation of Na⫹-currents could additionally cause a slowed central as well as peripheral conduction velocity and impair synaptic transmission, especially at higher firing frequencies of the presynaptic nerve (see Fig. 4). The hypothesis, that a regulation of Na⫹-currents could play a substantial role in the origin of the neurological symptoms resulting from thyroid dysfunction, has, however, so far not been elaborated in much detail. Two studies in the past indicated an increased rate of Na⫹ entry (Raskin and Fishman, 1966) as well as an increase in the intracellular brain Na⫹ concentration (Timiras et al., 1955) by thyroid hormone. Two other studies, using 3[H] saxitoxin binding, presented a first evidence for a regulation of Na⫹-channels by thyroid hormone in myotubes and skeletal muscle cells (Brodie and Sampson, 1989; Harrison and Clausen, 1998). Here we show, in extension of our first study on postnatal rat hippocampal cells (Potthoff and Dietzel, 1997), that T3 regulates Na⫹-currents with respect to K⫹-currents in cultured as well as acutely dissociated cortical neurons from neonatal rats. Furthermore, we now show that the regulation of the Na⫹-current density is large enough to induce changes in action potential shape and firing frequency.

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The results showing the most prominent effect on firing frequencies with higher stimulus intensities are in line with an investigation of Madeja (2000), who showed that, although considerable down-regulations in Na⫹-current density are needed to cause gross alterations in action potential shapes, smaller changes in Na⫹-current density induce an increase in the refractory period and a decrease in the firing frequency. In addition to the regulation of somatic Na⫹-channels shown here, a decrease in sodium channel density in the axons of peripheral nerves, for which only indirect evidence is so far available (Horsten and Boeles, 1949; Makii et al., 2002), could explain the decreased peripheral conduction velocities and the increased latencies of evoked potentials found in hypothyroidism (Bradley et al., 1961; O’Malley et al., 1985; De Vries et al., 1986; Laureau et al., 1987; Giroud et al., 1988; Norcross-Nechay et al., 1989; Albee et al., 1989). It could, furthermore, underlie the decreased amplitudes of evoked potentials observed in hypothyroidism (Bradley et al., 1961; Giroud et al., 1988; Huang et al., 1989; Albee et al., 1989) and the decreased amplitudes of peripheral compound action potentials found in hypothyroid rats which could not be explained on the basis of an impaired myelination (Quattrini et al., 1993). Likewise, a regulation of voltage-gated inward currents by thyroid hormone could contribute to the increased amplitudes of evoked potentials observed in hyperthyroidism (Short et al., 1968; Takahashi and Fujitani, 1970; Freeman and Sohmer, 1995). It has been suggested that in unmyelinated nerve there is an optimal density of sodium channels that ensures maximal neuronal conduction velocity (Hodgkin, 1975), beyond which no further increase or even a slowing of conduction velocity occurs. Hence an upregulation of sodium channels by thyroid hormone could possibly also explain the inconsistent findings concerning latencies of evoked visual potentials in hyperthyroidism, where some authors found decreases in latencies (Kopell et al., 1970) and others found only slight changes (Takahashi and Fujitani, 1970; Huang et al., 1989) or even increases with increases in thyroid hormone levels (Short et al., 1968). More detailed experiments are needed to resolve this issue completely. Thyroid hormone concentrations under experimental, physiological and pathophysiological conditions Unbound (free) T3 concentrations (fT3) in humans normally amount to 3.0⫾0.7 pM, while total protein bound serum concentrations (TT3) of 1.9⫾0.3 nM are found (Konno et al., 1987). In healthy rats, mice and guinea-pigs, likewise, fT3 levels of about 1–3 pM (Di Meo et al., 1994) and TT3 values of about 1 nM have been reported (Felzen et al., 1991; Di Meo et al., 1994; Shimoni et al., 1997; Knipper et al., 1999). In human hyperthyroidism fT3 values of 36.3⫾23.8 pM, concomitant with total T3-concentrations of 7.9⫾3.2 nM have been observed (Konno et al., 1987) but in some patients even extreme values of up to 30 nM TT3 and 120 pM fT3 have been measured (Hollander et al., 1972). To obtain clear results and to use concentrations consistent with other studies of thyroid hormone ac-

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tion in culture (see Experimental Procedures) the experiments on cultured cells were performed in the severely hyperthyroid range (30 nM, with a resulting fT3 ⬎40 pM). The experiments on acutely dissociated neurons were performed in a concentration range (0.9 to ⬎40 pM fT3) roughly comparable to that used in culture. In the acutely dissociated cells both, the downregulation of the Na⫹/K⫹-current ratio in hypothyroid rat pups as well as its upregulation in cells from hyperthyroid pups were statistically significant compared with currents from untreated controls. Hence a deviation from the physiological range may affect Na⫹-current density in both directions, consistent with clinical observations of some accelerated mental functions in hyperthyroidism and slowed functions in hypothyroidism. For example, the critical flicker fusion frequency, which is the minimal frequency at which the perception of light flashes turns into a continuous light signal is about 10% higher in hyperthyroid test persons (41– 48 Hz) than in euthyroid test persons (37 Hz; Beck, 1949). Ten percent to 20% decreased values of the critical flicker fusion frequency are found in hypothyroidism (Enzer et al., 1941; Levander and Rosenqvist, 1979). Together with our previous results which showed a statistically significant upregulation of Na⫹-current densities after preincubation with 5.2 nM T3 (about twice the physiological T3 concentration) our present results indicate that the physiological range is found somewhere in the middle of the dose-response curve for the Na⫹-current regulation by T3. To determine the exact position of the physiological range on the dose-response curve for the regulation of the Na⫹-current density by T3 more detailed experiments with a precise determination of the fT3-values relating to different TT3 concentrations in the bath solution (which will depend on the carrier proteins and T4 values present (Konno et al., 1987) will have to be performed. Molecular mechanisms of Naⴙ-current regulation by thyroid hormone Most actions of thyroid hormone are thought to be mediated via a regulation of protein synthesis driven by binding of T3 to several thyroid hormone receptors (Williams, 1994; Oppenheimer and Schwartz, 1997). So far, however, only a few genes have been shown to be direct targets of thyroid hormone in the brain (Thompson and Potter, 2000). In addition to its action via nuclear receptors possible direct membrane effects have, additionally, been reported (Davidoff and Ruskin, 1972; Martin et al., 1996). Hence the increased excitability observed after iontophoretically applied thyroid hormone in cat brain (Davidoff and Ruskin, 1972) as well as the bursting of Na⫹-channels in rat and rabbit heart cells following acute thyroid hormone application (Harris et al., 1991; Dudley and Baumgarten, 1993; Huang et al., 1999) represent possible membrane effects of thyroid hormone. Such effects could occur via direct effects on channel gating or influence Na⫹-currents via membrane receptors and second messenger cascades in the range of minutes (see Davis and Davis, 2002 for a review of nongenomic effects of thyroid hormone on the heart).

Most effects of thyroid hormone occur, however, on a larger time scale, more compatible with a regulation of gene expression via nuclear receptors (Oppenheimer and Schwartz, 1997). In agreement with this concept in skeletal myotubes (Brodie and Sampson, 1989) and muscle fibers (Harrison and Clausen, 1998) thyroid hormone has been shown to induce a delayed and protein synthesis dependent upregulation of [3H]-saxitoxin binding. We presently observed the T3-induced Na⫹-current regulation in T3-free recording solutions, which is in line with the assumption of a regulation via nuclear receptors. Interestingly, we observed a slightly larger regulation of Na⫹-currents in hippocampal as compared with cortical neurons. This finding corresponds to the observation of a more prominent expression of thyroid hormone receptor mRNAs in hippocampus than in cortex of rats (Bradley et al., 1989). Further experiments are needed to conclusively answer the question whether neuronal Na⫹-currents are regulated by thyroid hormone via nuclear receptors or whether membrane receptors are involved as well. Finally, the possibility remains that Na⫹-currents are regulated by thyroid hormone secondarily via an upregulation of growth factors, the synthesis of which has also been shown to be stimulated by thyroid hormone (Lindholm et al., 1994; Lu¨sse et al., 1998). Thus brain derived neurotrophic factor regulates Na⫹-currents in hippocampal progenitor cells (Sah et al., 1997) and nerve growth factor has been shown to differentially regulate Na⫹-channel expression in peripheral dorsal root ganglion cells (Omri and Meiri, 1990; Fjell et al., 1999), PC12 cells (Mandel et al., 1988) as well as neuroblastoma cells (Lesser and Lo, 1995). While NGF is mainly involved in the regulation of development, regeneration and sensitivity of peripheral nerves including sensation of pain (Gould et al., 2000; Waxman et al., 2000), thyroid hormone exerts more generalized effects on the CNS, influencing among others vigilance, memory and the ability to concentrate (Kudrjavcev, 1978; Swanson et al., 1981). Via a specific regulation of different subtypes of Na⫹-channels at different time scales the function of different types of neurons and the intensity of neuronal reactions could thus be adjusted to some extent by specific trophic or hormonal factors. Acknowledgements—This project was supported by the German “Hochschulsonderprogramm II” and by a graduate student fellowship of Nordrhein-Westfalen to G.H. We want to thank I. Grelle and Chr. Reiners for the determination of the fT3 concentrations in rat sera, Jens Meyer for building the amplifiers, Stefan Mann and Patrick Happel for helpful discussions and Rolf Heumann for continuous support.

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(Accepted 12 January 2004)