Vesicular tracking and stimulus-dependent modification of NET-1 rate in sympathetic nerve terminals

Julia Shanks1, Lauren Parker1, James Kennard1, Keith Brain2,1. 1Univerisity of Oxford, Oxford, United Kingdom, 2University of Birmingham, Birmingham, United Kingdom.

Many active processes exist within sympathetic nerve terminals which are difficult to observe dynamically in mature tissue preparations. We have demonstrated the use of a novel fluorescent substrate of the norepinepherine uptake transporter (NET) and the vesicular monoamine transporter (VMAT), called the Neurotransmitter Transporter Uptake Assay (NTUA; MDS Analytical Technologies), to measure NET activity in sympathetic terminals in the mouse vas deferens (Parker et al., 2009). We have used this technique to investigate the frequency dependent effect of field stimulation on NET uptake rate and the rate at which neurotransmitter vesicles exchange between nerve terminal varicosities.

Electrical field stimulation caused a frequency dependent decrease in the rate of NTUA uptake, levelling to a non-zero baseline at frequencies equal to or greater than 4 Hz. Exogenous application of a high concentration of noradrenaline (10 μM) had no effect on the rate of NTUA uptake (rate change of 5.0 ± 3.5 %/min; P > 0.05, n = 31 terminals from 3 animals) indicating that the results observed were not due to increased competition for uptake by junctional noradrenaline. Therefore the frequency-dependent effects observed are due to a decrease in NET rate.

Fluorescence recovery after photo bleaching (FRAP) of a targeted varicosity (n = 6) allowed the rate of vesicle exchange along the nerve terminal to be measured. Fluorescence recovery was observed to occur in a punctate manner, consistent with movement of NTUA containing vesicles between varicosities. Rate of fluorescent recovery was slow (time to recover to within 1/e of the control, median 120 min; range 107-250 min) demonstrating a slow rate of vesicle exchange between varicosities in such sympathetic terminals.

These results demonstrate the use of NTUA to show how frequency of nerve stimulation can affect NET transporter rate. Since NTUA is also a substrate for VMAT, we have also demonstrated its use to measure vesicle movement along sympathetic nerve terminals for the first time.

Parker LK, Shanks JA, Kennard JAG, Brain KL (2009). Dynamic monitoring of NET-1 activity in mature murine sympathetic terminals using a fluorescent substrate. Br J Pharmacol, in the press.