Circ Res 94: 1500C1506, 2004. 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 0.01) without changing cell capacitance (control: 3.2 0.3 pF; = 21; T4: 3.2 0.2 pF; = 22; = 0.87). In contrast, chronic application of the thyroid hormone blocker tetrac reduced peak 0.05) (Fig. 1= 17; = 0.89). Chronic application of 30 nM T3 did not significantly alter peak = 18), T4 (?; = 8), T3 (3,3,5-triiodo-l-thyronine, ?; = 8), T4 + tetrac (?; = 6), and tetrac (; = 8) media. Chronic exposures to T4 but not T3 increased peak 0.01). Effects of T4 were prevented by coapplication of the antagonist tetrac. Moreover, tetrac alone reduced 0.05). To test whether the chronic T4 effects were specific to and and relationships for both control (?; = 4) and T4 (; = 4) treated RB recordings. The leak conductance, evaluated from the slope of the curve, was unaffected by T4 treatment. = 5) had no effect on steady-state = 17). We next examined whether T4 modulated sodium maximal conductance ( 0.05), whereas chronic incubation in 30 nM T3 had no significant effect on sodium and = 12) and decreased by either 30 nM T4 + 10 M tetrac (= 6) or 10 M tetrac alone (= 9) compared with controls (= 18) at 50C55 hpf (* 0.05, ANOVA). 0.05) (Fig. 3touch-insensitive mutant has shown that reduction of RB 0.0005 vs. control; ** 0.0001 vs. control). We tested the touch responses of spontaneously swimming tetrac-treated embryos. Embryos were tested for normal swimming behavior in response to touch, an abnormal response to touch such as a single trunk bend without swimming movement, or no response to touch. Normal responses indicate propagation of an action potential from the RB cell Xanthone (Genicide) to the ipsilateral Mauthner neuron, which in turn initiates a swimming pattern among contralateral motoneurons, whereas an abnormal response may suggest activation of local interneuron circuits by RB cells, but not activation of the Mauthner neuron (Fig. 4 0.01) (Fig. 4 0.05) (Fig. 5= 10) compared with control (= 31) and sham (= 9) treatments. Supplementing embryos with 30 nM T4 (= 4) immediately after surgery rescued the effects of yolkectomy (* 0.05, ANOVA). = 4) and sham yolkectomy (= 5) had no effect on = 17). = 35) relative to controls (= 8). The effect of yolkectomy, however, was partially rescued by addition of 30 nM T4 to embryos after yolkectomy (= 35), as indicated by the increase in the percentage of trials showing normal responses (* 0.01; ** 0.001). Consistent with the reduction in RB 0.001) (Fig. 5 0.001), indicating Xanthone (Genicide) that some of the yolkectomy effects were due to thyroid hormone deficiency. However, yolkectomized embryos that received T4 treatment still showed a decrease in normal touch responses versus control ( Xanthone (Genicide) 0.01), suggesting that some of the effects of yolk sac reduction on behavior were independent of T4. Overall, the results indicate that endogenous thyroid hormone acts in vivo to maintain a sufficient 0.05). In contrast, addition of 1 1 to 100 nM T3 did not significantly affect = 4; 1 nM, = 4; 10 nM, = 6; 30 Rabbit Polyclonal to CPZ nM, = 11; 100 nM, = 6; 1 M, = 3) was not mimicked by T3 () (1 nM, = 4; 30 nM, = 3; 100 nM, = 5; 300 nM, = 2; 1 M, = 5; 10 M, = 3), over a wide range of concentrations. Acute application of T4 to RBs increased peak = 9) (* 0.05, ANOVA). Next we tested whether the thyroid hormone antagonist tetrac blocked the acute effects of T4. Coapplication of tetrac prevented the acute effect of T4 Xanthone (Genicide) on = 11) within 5 min compared with controls (= 9) at 50C55 hpf. In contrast, acute T3 did not change = 3). Acute T4 effects were blocked by coapplication of 10 M tetrac (= 3), chronic incubation in 30 nM T4 (= 7), or injection of 50 g/mL LM609 but not vehicle (H2O) (= 3) (* 0.05, ANOVA). Xanthone (Genicide) = 5) did not affect = 6). 0.01 vs. control; ** 0.001 vs. control). T4 modulates INa via integrin.