Evoked local field potential recorded from lymph heart before and after addition of carbachol followed by atropine in the presence of carbachol.

<p>A) Representative trace of evoked local field potential (LFP) obtained from control, in the presence of carbachol (10μM) B) and after adding atropine (5μM) in the presence of carbachol C). D) Bar charts show average peak to peak of evoked LFP amplitude calculated from control, in the presence of carbachol and after adding atropine in the presence of carbachol (n = 9). The data for evoked LFP in the presence of carbachol and the data for evoked LFP of atropine in the presence of carbachol was normalised to data obtained from control. After adding carbachol the average peak to peak of evoked <b>LFP</b> amplitude was significantly reduced (*** p<0.001) compared to the control. On addition of atropine in the presence of carbachol the effects were significantly reversed (*** p<0.001). Error bars represent mean ± SEM (n = 9). E) Bar chart shows average peak to peak of evoked LFP amplitude calculated from control, in the presence of TTX (n = 3). The data for evoked LFP in the presence of TTX was normalised to data obtained from control. After adding TTX the average peak to peak of evoked LFP was reduced compared to control however it was not statistically significant (p>0.05). Error bars represent mean ± SEM.</p

Expression of HCN1 and HCN4 at HH36.

<p>Whole mount in-situ hybridisation showing expression of <i>HCN1</i> and <i>HCN4</i> in avian lymph heart at HH36 as indicated by red arrow. The expression of these channels was not detected in other stages.</p

Evoked local field potential recorded from lymph heart before and after addition of cyclopiazonic acid followed by calcium.

<p>A) Representative trace of evoked local field potential (LFP) obtained from control, in the presence of CPA (10μM) B) and after adding Ca²⁺ (2mM) C). D) Bar charts show average peak to peak of evoked LFP amplitude calculated from control, in the presence of CPA and after adding Ca²⁺ (n = 13). The data for the evoked LFP in the presence of CPA and the data for the evoked LFP of Ca²⁺ was normalised to data obtained from control. After adding CPA the average peak to peak of evoked LFP amplitude was significantly reduced (* p<0.05) compared to control. On addition of Ca²⁺ the average peak to peak of evoked LFP amplitude significantly increased compared to CPA (*** p<0.001) and control (* p<0.05). Error bars represent mean ± SEM (n = 13).</p

Evoked local potential recorded from lymph heart before and after addition of ZD7288 and effect of ZD7288 on lymph heart beating.

<p>A) Representative trace of evoked local field potential (LFP) obtained from control and in the presence of ZD7288 (20μM) (B). C) Bar charts show average peak to peak of evoked LFP amplitude calculated from control and after adding ZD7288 (n = 11). The data for the evoked LFP in the presence of ZD7288 was normalised to data obtained from control. After adding ZD7288 the average peak to peak of evoked LFP amplitude was significantly reduced (*** p<0.001) compared to the control. Error bars represent mean ± SEM (n = 11). D) Representative trace obtained by plotting z-axis profile of lymph heart beating. The arrow on the left indicates the time at which ZD7228 is added. The lymph heart beating declined with time and eventually stopped beating completely as indicated on the trace.</p

Summary of the genes regulated during lymph heart development and involvement of ion channels in excitability and rhythmicity of the lymph heart.

<p>The lymph heart predominantly expresses skeletal muscle markers and their expression is evident as early as HH30 and decline<b>s</b> by HH38. By HH36, the lymph heart contracts rhythmically and the basis for their underlying rhythmicity is most likely mediated by HCN channels while their excitation and contraction coupling is facilitated by cholinergic and L-type Ca²⁺ channels.</p

Expression of Prox-1, and Cav1.1 during lymph heart development.

<p>Whole mount in-situ hybridisation showing developmental expression of <i>Prox-1</i> (A1-A4) and <i>Cav1</i>.<i>1</i> (B1-B4) in the lymph heart as indicated by red arrow. The expression of <i>Prox-1</i> and <i>Cav1</i>.<i>1</i> was detected as early as HH30 and declined by HH38.</p

Expression of cadherins during lymph heart development.

<p>Whole mount in-situ hybridisation showing developmental expression of <i>M-cadherin</i> (A1-A4), <i>N-cadherin</i> (B1-B4), <i>R-cadherin</i> (C1-C4) and <i>T-cadherin</i> (D1-D4) in the lymph heart as indicated by red arrow. The expression of <i>M</i>, <i>N</i>, and <i>R</i>, and <i>T-cadherin</i> was detected as early as HH30 and only <i>M-cadherin</i> was expressed at HH38.</p

Expression of skeletal muscle markers during development of lymph heart.

<p>Whole mount in-situ hybridisation showing developmental expression of <i>Pax-7</i> (A1-A4), <i>MyoD</i> (B1-B4), <i>Myogenin</i> (C1-C4) and Engrailed-1 (<i>En-1</i>) (D1-D4) in the lymph heart as indicated by red arrow. The expression of <i>Pax-7</i>, <i>MyoD</i>, <i>Myogenin and En-1</i> was detected as early as HH30 and declined by HH38.</p