Proceedings of The Physiological Society
Trinity College Dublin (2003) J Physiol 551P, PC41
Kv3.4 potassium channel subunit immunoreactivity in presynaptic terminals in the brainstem and spinal cord of rats
R.E. Brooke, S.A. Deuchars and J. Deuchars
School of Biomedical Sciences, University of Leeds LS2 9NQ, UK
Voltage gated potassium channel subunits of the Kv3 subfamily (Kv3.1-Kv3.4) play a vital role in action potential repolarisation in neurones (Rudy & McBain, 2001). Whereas Kv3.1-Kv3.3 appear to encode delayed rectifier currents which facilitate fast firing, the role of Kv3.4 subunits in neurones is less clear. Cellular and sub-cellular differences in the pattern of ion channel expression are important in defining specific roles of channels and since little is known regarding the possible presence of this subunit in the spinal cord and medulla, we used immunohistochemistry to study its distribution in these regions.
Rats (100-200 g, n = 5) were injected intraperitoneally with 0.1ml 1 % Fluorogold (Fluorochrome Inc.) and 7 days later were humanely killed by Sagatal (100 mg kg-1 I.P.) and perfused transcardially with 4 % paraformaldehyde-0.1-0.5 % glutaraldehyde. Slices (50 µm) of medulla and thoracic spinal cord were cut and incubated in anti-Kv3.4 antibody (Alomone; 1:1K) followed by a Cy3-conjugated secondary antibody. Sections were also incubated in antibodies raised against SV2, VGluT1, VGluT2 and GlyT2 (Iowa Uni Hybridoma Bank, 1:500; Chemicon, 1:20K, 1:5K and 1:5K, respectively), which were visualised using a biotinylated conjugated secondary antibody and Streptavidin Alexa.
Kv3.4 immunoreactivity was observed in punctate structures throughout the spinal cord and medulla. Particularly dense labelling was observed in regions involved in autonomic control such as the intermediolateral cell column (IML) in the spinal cord and the dorsal vagal nucleus (DVN) in the medulla. These punctate structures, suggestive of terminals, closely apposed Fluorogold labelled autonomic neurones and motoneurones. Co-localisation of Kv3.4-IR with the synaptic vesicle protein, SV2, provided confirmation of presynaptic labelling. Furthermore, co-localisation of Kv3.4-IR with the glutamate vesicle transporters, VGluT1 and VGluT2, and the glycine transporter, GlyT2, demonstrated that these subunits are present in both excitatory and inhibitory presynaptic terminals.
This study indicates that Kv3.4 subunits are found in presynaptic terminals throughout the spinal cord and brainstem, with strong labelling in regions involved in autonomic control. These results suggest that Kv3.4-containing channels, probably by modulating the shape of invading action potentials, regulate neurotransmitter release from excitatory and inhibitory terminals.
This work was funded by the British Heart Foundation
Where applicable, experiments conform with Society ethical requirements