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Department of Physiology and Biophysics, Medical Scientist Training Program, Neuroscience Program, University of Colorado Denver, Anschutz Medical Campus, Aurora, Colorado
Submitted 24 July 2008; accepted in final form 11 September 2008
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ABSTRACT |
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Vβ3, occluded the rapid effect of T4, implicating a specific integrin dimer as a T4 receptor. Chronic application of either tetrac or LM609 significantly reduced sodium conductance, demonstrating an in vivo requirement for T4-integrin regulation of INa. Further, removing endogenous T4 levels via yolkectomy reduced sodium conductance, an effect that was partially rescued by T4 supplementation following surgery. Because RBs mediate the embryonic touch response, we tested for behavioral effects. Tetrac and LM609 significantly reduced the percentage of touch trials eliciting a normal touch response. T4's rapid effect on RB INa highlights the importance of embryonic T4 availability and nongenomic T4 signaling. |
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INTRODUCTION |
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; Contempre et al. 1993; Ferreiro et al. 1988; James et al. 2007; Vulsma et al. 1989). For example, lack of maternal thyroid hormone exposure during embryonic development causes irreversible mental retardation (Hetzel 2000) and maternal hypothyroidism results in developmental delays and behavioral deficits after birth (Francis and Riley 1987; Mirabella et al. 2000; Pacaud et al. 1995; Rovet and Hepworth 2001; Smit et al. 2000). Conversely, maternal hyperthyroidism poses risks to fetal development by significantly increasing miscarriage rates (Anselmo et al. 2004; Kriplani et al. 1994). The clinical symptoms observed following altered maternal thyroid hormone states during embryonic stages indicate that thyroid hormones regulate neuronal properties during early development.
Previous data show that thyroid hormones modulate voltage-gated sodium current (INa) in neonatal cardiac muscle (Huang et al. 1999) and neurons (Potthoff and Dietzel 1997). However, the effects of thyroid hormone on embryonic neurons are not well studied. INa not only underlies rapid signaling in neurons but also regulates many aspects of neurodevelopment, including programmed cell death (Svoboda et al. 2001), axonal morphology (Pineda et al. 2006), and behavior (Ribera and Nüsslein-Volhard 1998). If thyroid hormone regulated voltage-gated sodium channel function during embryonic stages, then rapid signaling as well as neurodevelopment would be altered by thyroid disorders.
Conventional thyroid hormone action involves triiodothyronine (T3) binding to nuclear thyroid hormone receptors and modulation of gene expression, producing cellular effects after considerable delay. However, emerging evidence supports an alternate signaling pathway that requires plasma membrane receptors, occurs rapidly within minutes, and shows increased selectivity for thyroxine (T4), the T3 precursor (Davis et al. 2004). In vitro studies with cultured fibroblasts or chick chorioallantoic membranes indicate that nongenomic effects require T4 interaction with an Vβ3 integrin to initiate rapid effects (Bergh et al. 2005; Mousa et al. 2006). The Vβ3 integrin belongs to the Arg-Gly-Asp (RGD)–type integrin family, which influences zebrafish neurodevelopment (Becker et al. 2003). In addition, the RGD protein-recognition site of Vβ3 binds T4 (Davis et al. 2005). Whether T4 integrin-dependent mechanisms also play a role in vivo during embryonic neurodevelopment is unknown.
The developing zebrafish (Danio rerio) embryo provides a model to test for in vivo roles of thyroid hormone in regulation of neuronal function during embryogenesis. We assayed physiological function of embryonic Rohon–Beard cells (RBs) using both whole cell voltage-clamp and behavioral assays. We focused on INa because of the important roles this current plays in neuronal signaling (Pineda et al. 2006; Svoboda et al. 2001). Moreover, the macho mutant has demonstrated that the embryonic touch response requires a critical RB INa density to initiate swimming behavior (Granato et al. 1996; Ribera and Nüsslein-Volhard 1998), allowing for a behavioral assay of RB INa modulation. In addition, the zebrafish embryo allows manipulation of thyroid hormone signaling in vivo by adding exogenous or decreasing endogenous T4 levels and by blocking endogenous T4 receptors. Using these approaches, we obtained evidence for an integrin-dependent nongenomic T4 mechanism in vivo that rapidly modulates INa in embryonic sensory neurons.
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METHODS |
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Zebrafish (Danio rerio) adults were bred according to guidelines outlined in The Zebrafish Book (Westerfield 1995). Embryos were incubated at 28.5°C in embryo media (EM: 130 mM NaCl, 0.5 mM KCl, 0.02 mM Na2HPO4, 0.04 mM KH2PO4, 1.3 mM CaCl2, 1.0 mM MgSO4, 0.4 mM NaH2CO3) and staged according to external morphology (Kimmel et al. 1995).
Electrophysiology
Whole cell voltage-clamp recordings were obtained from zebrafish spinal cord RBs at 50- to 55-h postfertilization (hpf) (Pineda et al. 2005; Ribera and Nüsslein-Volhard 1998). Zebrafish were immobilized in Ringer solution [(in mM):145 NaCl, 3 KCl, 1.8 CaCl2, and 10 HEPES, pH 7.2] containing 0.02% tricaine (Sigma) and glued to glass coverslips with Vetbond (3M, St. Paul, MN) tissue adhesive. Glass dissecting needles sufficed for removal of the skin, thus avoiding use of proteolytic enzymes. After dissection, the preparation was washed with 
40 mL extracellular recording solution for 15 min, providing a semiintact preparation for recordings. We used a reduced extracellular sodium bath solution [(in mM): 30 NaCl, 97 N-methyl glucamine, 20 tetraethylammonium, 3 KCl, 2 CoCl2, and 10 HEPES] to limit INa amplitudes and reduce potential series resistance voltage errors (Pineda et al. 2005). Glass electrodes (2.0–3.5 M
) were filled with solution containing (in mM): 10 NaCl, 135 CsCl, 10 EGTA, and 10 HEPES. We subtracted passive leak currents and capacitive transients from recordings of voltage-gated sodium and potassium currents using a P/8 protocol. Data were recorded using an Axopatch 200B amplifier (Axon Instruments, Foster City, CA) and data were analyzed with Clampfit8 (Axon Instruments) and Origin software (OriginLab, Northhampton, MA). Results are presented as means ± SE. Statistical analysis was performed with Origin v7.0 software (OriginLab). Except for the behavioral data presented in the following text, statistical comparisons of means were performed using unpaired two-tailed t-tests or one-way ANOVAs with Bonferroni correction for multiple comparisons.
Hormone and drug application
Solutions of T4 (3,3',5,5'-tetraiodo-L-thyronine; Sigma), T3 (3,3',5-triiodoLthyronine; Sigma), and tetrac (3,3',5,5'-tetraiodothyroacetic acid; Sigma) were diluted to final concentrations in solutions of embryo media for chronic recordings and extracellular recording solution for acute recordings. During chronic application, hormone solutions were replaced daily to minimize deiodination effects. Tetrac (10 µM) was applied to embryos by addition to embryo media. The LM609 antibody (Millipore, Danvers, MA) was microinjected directly into the yolk sac of 24-hpf embryos. LM609 (50 µg/mL) was diluted with H2O and 1% Fast Green dye to visually confirm distribution of microinjections throughout the zebrafish embryo. Vehicle controls consisted of 1% Fast Green injections.
Behavioral studies
We assayed touch sensitivity of 48-hpf embryos as described by Pineda et al. (2005). We performed touch sensitivity assays with the observer impartial to the treatment groups. To focus on sensory versus motor function, we verified that embryos could swim spontaneously with a normal pattern. Next, we gave each embryo ten touch trials with a 3-s rest between trials; each trial entailed gentle application of a metal probe to the dorsal trunk. For each trial, the response outcome was scored as none, abnormal (e.g., segmentally restricted trunk bend or twitching movement), or normal (i.e., swimming response). Results are presented as histograms that display the percentage of trials generating each response outcome for each treatment group. For each outcome, we compared different treatment groups with nonparametric statistical tests (Mann–Whitney or Kruskal–Wallis followed by Dunn's test for multiple comparisons) using InStat3 software (GraphPad) and present data as means ± SE.
Yolkectomy
To reduce endogenous T4 stores, we partially removed (50%) the yolk sac by suction using a mouth pipette from 23- to 25-hpf wildtype embryos. Following surgery, embryos were placed in either normal EM or EM supplemented with 30 nM T4 for recovery experiments and returned to a 28.5°C incubator. Sham yolkectomies consisted of yolk sac puncture only without suction.
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RESULTS |
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In initial experiments, we raised zebrafish embryos beginning with the first hpf in embryo media containing either 30 nM T4, 30 nM T3, or no hormone. To test for thyroid hormone effects on RB INa in 50- to 55-hpf embryos, we elicited sodium currents with depolarizing pulses (Fig. 1A). Chronic T4 exposure increased peak INa by 46% over control (P < 0.01) without changing cell capacitance (control: 3.2 ± 0.3 pF; n = 21; T4: 3.2 ± 0.2 pF; n = 22; P = 0.87). In contrast, chronic application of the thyroid hormone blocker tetrac reduced peak INa by 37% compared with control (P < 0.05) (Fig. 1B), demonstrating that T4 interactions with a thyroid hormone receptor modulated INa. Tetrac application did not significantly affect cell capacitance compared with controls (tetrac 3.3 ± 0.2 pF; n = 17; P = 0.89). Chronic application of 30 nM T3 did not significantly alter peak INa compared with controls. The current–voltage plots demonstrated that peak amplitudes for controls occurred near 0 mV and reversal potentials averaged near +22 mV, close to the calculated reversal potential of +27.7 mV. In response to T4 incubation, INa amplitude increased at all test potentials without changing the reversal potential (Fig. 1C).
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The data presented earlier indicate that exogenous T4 modulates RB INa. However, application of exogenous hormone does not address whether endogenous T4 regulates RB INa in vivo. Maternally derived stores present in the embryonic yolk sac provide T4 to the embryo prior to function of the zebrafish thyroid gland at 72 hpf (Brown 1997). We pharmacologically blocked endogenous T4 by incubating embryos in 10 µM tetrac chronically and recorded RB INa at 50–55 hpf. Incubation in 10 µM tetrac significantly reduced sodium Gmax by 46% (P < 0.05) (Fig. 3A), consistent with blockade of endogenous T4 and an in vivo role for T4 INa regulation.
Application of tactile stimuli to RB cell free nerve endings in the skin triggers the touch response. After tactile stimulation RBs fire an action potential that recruits involvement of many cell types (neurons, muscle) and synapses (Fig. 4A). Study of the macho touch-insensitive mutant has shown that reduction of RB INa density can prevent the behavioral response to touch without affecting spontaneous swimming behavior (Granato et al. 1996; Ribera and Nüsslein-Volhard 1998). Because tetrac treatment produced such a large reduction in the RB sodium Gmax we hypothesized that RBs might not be able to mediate the touch response (Fig. 4A).
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As an independent test of the in vivo requirement for endogenous T4, we surgically removed about 50% of the yolk sac, the location of maternal deposits of thyroid hormone present prior to function of the embryo's thyroid gland (Fig. 5A) (Tagawa et al. 1990; Walpita et al. 2007). Compared with control embryos and sham yolkectomies we found that 50% yolkectomy reduced RB sodium Gmax by 41% (P < 0.05) (Fig. 5B). Addition of 30 nM T4 to the embryo media immediately following a 50% yolkectomy rescued sodium Gmax to control values (Fig. 5B). Despite rescue by T4 of yolkectomy effects on INa, other developmental signals within the yolk may have had general nonspecific effects. To control for nonspecific effects on ion currents, we tested whether yolkectomy altered IKv. In contrast to INa, RB IKv steady-state amplitude was not significantly different from controls in 50% yolkectomized embryos (Fig. 5C).
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T4 rapidly modulates RB INa
Because the nongenomic thyroid hormone pathway displays a faster time course than that of the conventional genomic pathway (Davis and Davis 1996), we tested whether T4 would rapidly regulate INa. We acutely added either T4 or T3 to the bath solution and recorded effects on peak INa amplitudes within 5 min of application (Fig. 6). We present the data as the percentage change in peak INa amplitude that occurred within 5 min of drug application. Positive values indicate that the drug treatment rapidly increased INa amplitude, whereas negative values reflect a decrease in peak current over time. We found that acute T4 application rapidly increased RB INa peak amplitude by 39% (Fig. 6A). The rapid effect of T4 on INa amplitude permitted a dose–response analysis with T4 concentrations varying between 100 pM and 1 µM (Fig. 6B). Because T3 activates the nongenomic pathway in some systems (Bergh et al. 2005; Sarkar et al. 2006), we also tested for T3 effects over a broad concentration range (1 nM to 10 µM). Addition of 30 nM, 100 nM, and 1 µM T4 significantly increased INa peak amplitude over changes in control amplitudes (P < 0.05). In contrast, addition of 1 to 100 nM T3 did not significantly affect INa peak amplitude. Overall, the results demonstrate rapid modulation of RB INa by T4 but not T3.
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Vβ3
The rapid and selective effect of T4 versus T3 on INa density suggested a nontraditional thyroid hormone signaling pathway. Because the RGD-type integrin dimer Vβ3 has been implicated in vitro as a plasma membrane receptor for T4 signaling (Bergh et al. 2005), we tested whether the function-blocking Vβ3 antibody LM609 (Cheresh and Spiro 1987) could affect RB cell sodium current and embryonic behavior. We injected 50 µg/mL LM609 into 24-hpf zebrafish embryos and recorded RB INa at 50–55 hpf. LM609 injection significantly reduced RB peak INa amplitudes by 46% (557 ± 32 vs. 303 ± 43 pA; n = 13 and 9, control and LM609 treated, respectively; P < 0.005). Further, LM609 significantly reduced the percentage of touch response trials eliciting a normal response (Fig. 7C; P < 0.001), consistent with Vβ3 serving as a T4 receptor.
If the acute effects of T4 were mediated by Vβ3, then LM609 injection would be expected to block acute T4 effects on INa. We found that LM609 injection prior to acute T4 application occluded the rapid increase in RB INa amplitude produced by T4. In contrast, T4 elicited a rapid increase in INa amplitude in RBs of control vehicle-only injected embryos (Fig. 7A). These findings suggest that the Vβ3 integrin serves as a mediator for T4's rapid modulation of RB cell INa amplitude.
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DISCUSSION |
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Vβ3 integrin thyroid hormone receptor (Bergh et al. 2005) led us to test for nongenomic mechanisms during embryonic stages. Our results indicate that T4 rapidly modulates sensory neuron INa in embryonic zebrafish. The time course of T4 action is not consistent with genomic mechanisms. Moreover, we find that integrin Vβ3 is required for T4's rapid modulation of embryonic RB INa.
In both humans and zebrafish, the embryo does not begin to synthesize thyroid hormone until late gestation or larval stages, respectively. Consequently, in both species, maternal thyroid hormone provides the early embryo with access to thyroid hormone stores. We found that T4 initiated a rapid signaling pathway at concentrations 30 nM, a range similar to maternal contributions of total thyroid hormone during fetal development (30–70 nM; Vulsma et al. 1989). These considerations underscore the importance of maintaining maternal euthyroid status and placental function.
Our data show changes in both RB sodium current density and touch sensitivity in response to T4. All treatments that significantly reduced Gmax also produced a significant increase in the percentage of trials eliciting no touch response (Figs. 4, 5, and 7). This high level of agreement was not expected because other neurons, in addition to RBs, play a role in generation of the touch response (Fig. 4A). Further, T4 may affect other excitable membrane properties, even though we did not observe changes in voltage-gated or leak potassium currents. Nonetheless, the strong relationship between RB INa amplitude and touch sensitivity was observed regardless of whether we reduced T4-integrin signaling by reducing the effective levels of T4 (tetrac, yolkectomy) or blocking the novel T4 plasma membrane integrin receptor (LM609). In this regard, we obtained similar but slightly less robust results with echistatin, a blocker of the RGD-type integrin dimer family, of which Vβ3 is a member (data not shown). Taken together, the data suggest a strong relationship between RB INa amplitude and the touch response. However, because T4 may have undiscovered effects on other conductances or cells, it is not possible to conclude that T4's effect on touch response behavior is due solely to reduction of RB INa amplitude.
Our data implicate maternally provided T4 as an important regulator of RB INa. Reducing endogenous stores of T4 from the yolk sac significantly reduced peak sodium current and the percentage of touch response trials eliciting a normal response. Addition of 30 nM T4 to yolkectomized embryos immediately after surgery completely recovered effects on peak sodium current density and partially rescued the behavioral effects. Although the behavioral rescue promoted by T4 supplementation was statistically significant (Fig. 5D; P < 0.001), the percentage of normal touch responses was still reduced compared with that of control. The partial recovery suggests that the embryonic yolk sac houses factors other than T4 that affect the touch response. Overall, the data indicate that endogenous T4 stores play a role in maintaining RB cell INa and exert an influence on embryonic touch response behavior.
The touch response allows zebrafish embryos to flee from predators and thereby promotes survival. In addition to maintaining an appropriate physiological RB INa density, T4 modulated the embryonic touch response. The touch response swim reflex was compromised by either reducing thyroid hormone concentrations (surgically or pharmacologically) or blocking function of the Vβ3 integrin. We interpret the effects of T4 on behavior as a consequence of T4's effects on RB INa density. However, it is possible that T4 reduction, or Vβ3 blockade, had additional effects on other elements of the circuit mediating the touch response (Fig. 4A). Nevertheless, the results demonstrate that blockade of T4-Vβ3 signaling influenced a behavior critical for embryonic zebrafish survival.
Previous work has demonstrated that two different sodium channel -subunit isotypes, nav1.6 and nav1.1L, underlie RB INa (Pineda et al. 2005): nav1.6 channels carry the majority of RB INa, whereas nav1.1L channels carry a much smaller fraction. Further, nav1.6 channels pass current that reaches peak amplitude more rapidly than nav1.1L channels can. Our studies did not address whether T4 affects current in both channel types or in only one. Interestingly, although not studied in detail here, we observed that T4 consistently reduced time to peak INa (e.g., Fig. 1B). Due to the substantial and consistent increase in INa density and possible effects on kinetics, the data suggest that T4 may have selective effects on nav1.6 channels. Future studies will address this possibility.
In addition to the specific effects INa has on electrical excitability, INa also influences other aspects of neuronal differentiation such as axon morphology and cell death (Pineda et al. 2006; Svoboda et al. 2001). Disruption of maternal thyroid hormone status during early development would potentially alter INa and its effects on nervous system development. The long-term consequences of altered nongenomic T4 signaling during embryonic stages are unknown.
Our studies have focused on thyroid hormone action during embryonic development. However, thyroid hormone regulation of ion currents has been reported during other developmental stages through both conventional and rapid time courses (Huang et al. 1999; Potthoff and Dietzel 1997; Wang et al. 2003; Zinman et al. 2006). Whether T4's rapid regulation of INa via integrin Vβ3 is limited to early development or is cell specific to RBs is unknown. Nonetheless, our findings raise the possibility that rapid T4 signaling mechanisms and effects on INa could contribute to nervous system deficits associated with altered thyroid hormone status. Altogether, the identification of rapid thyroid hormone effects on neurons and the involvement of integrin Vβ3 in acute INa regulation support a nongenomic mechanism of action for T4 during embryonic stages and provide new insights into thyroid hormone mechanisms in the embryonic nervous system.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Address for reprint requests and other correspondence: M. A. Yonkers, Department of Physiology and Biophysics, University of Colorado Denver at AMC, RC-1 North Tower, Room 7403A, PO Box 6511, Mail Stop F8307, Aurora, CO 80045 (E-mail: marc.yonkers{at}uchsc.edu)
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ABSTRACT
INTRODUCTION
; n = 18), T4 (
; n = 8), T3 (3,3',5-triiodo-L-thyronine, 
; n = 8), T4 + tetrac (
; n = 6), and tetrac (
; n = 8) media. Chronic exposures to T4 but not T3 increased peak INa amplitude vs. control, respectively (P < 0.01). Effects of T4 were prevented by coapplication of the antagonist tetrac. Moreover, tetrac alone reduced INa amplitude, suggesting in vivo modulation of INa by endogenous T4 (P < 0.05).
; n = 4) treated RB recordings. The leak conductance, evaluated from the slope of the I–V curve, was unaffected by T4 treatment. C: potassium currents were elicited by voltage steps from –70 to +30 mV for 30 ms from a holding potential of –80 mV. D: steady-state IKv was measured as the mean current elicited by +30 mV for the last 5 ms of depolarization. Mean current was normalized for cell size and T4 treatment did not alter cell capacitance while recording IKv. Chronic incubation of 30 nM T4 (n = 5) had no effect on steady-state IKv compared with controls (n = 17).
) (1 nM, n = 4; 30 nM, n = 3; 100 nM, n = 5; 300 nM, n = 2; 1 µM, n = 5; 10 µM, n = 3), over a wide range of concentrations. Acute application of T4 to RBs increased peak INa at concentrations of 