Vesicle-associated proteins and P2X receptor clusters at single sympathetic varicosities in mouse vas deferens

1. Antibodies against vesicle-associated proteins of the SNARE complex (syntaxin (AbS), SNAP 25 (AbS25), synaptobrevin (VAMP; AbV) and the alpha 1B subunit of calcium channels (Ab alpha(1B)) were located with respect to sympathetic varicosities (labelled with the ubiquitous vesicle proteoglycan antibody AbSV2) and to clusters of P2X receptor subunits (labelled with antibodies AbP2X(1) to AbP2X(6)). In addition, these receptor clusters were located with respect to Schwann cells labelled with antibodies to S100 (AbS100). 2. The spatial relation between proteins of the SNARE complex and calcium channels was determined. AbS25 patches ranged from 250-500 nm in size and were often colocalised with smaller AbS patches (250-350 nm). Ab alpha(1B) patches (300-700 nm diameter) were always coincidental with AbS patches. AbV patches (400-1000 nm in diameter) also coincided with AbS patches. 3. The spatial relation between different P2X subunit clusters and varicosities labelled with AbSV2 was ascertained. Large (500-700 nm diameter) AbP2X(1) receptor clusters were found colocalised with many (91%) AbSV2 labelled varicosities, although small diameter (250-350 nm) AbP2X(1) clusters occurred at random over the muscle. Small AbP2X(2) clusters were found uniquely in the vicinity of AbSV2 labelled varicosities, but were not entirely coincidental with these. Small AbP2X(3) receptor clusters were found in close association with AbSV2 labelled nerves. Small diameter AbP2X(4) clusters (250-350 nm) were found throughout the muscle with some of these coincidental with AbSV2 labelling. Small diameter AbP2X(5) (250-350 nm) cluster labelling was found in juxtaposition to strings of AbSV2 labelled varicosities but were not coincidental with these. Small (250-350 nm) diameter AbP2X(6) clusters were also found in close juxtaposition to AbSV2 labelled nerves. 4. The spatial relation between different P2X subtype clusters and Schwann cells labelled with AbS100 was examined. Both AbP2X(1) and AbP2X(3) receptor clusters were found in close apposition with AbS100, with clusters of the former sometimes coincidental with patches of the latter. On the other hand AbP2X(2) was found in association with AbS100 at low levels while AbP2X(4) labelling was generally not coincidental with AbS100. AbP2X(5) and AbP2X(6) labelling was often colocalised with AbS100 labelling. 5. The spatial relation between proteins of the SNARE complex and P2X(1) receptors was determined. Large AbP2X(1) clusters were often found apposed by AbS, AbV and Ab alpha(1)B labelled patches. 6. Destruction of the sympathetic varicosities with 6-hydroxydopamine led to the virtual disappearance of AbP2X(2) labelling, but to a large increase in the number of small AbP2X(1) receptor clusters and a reduction in the number of large AbP2X(1) clusters. AbS100 Schwann cell labelling was largely unaffected. 7. These observations are interpreted as showing that most terminal sympathetic varicosities possess active zones about 250-700 nm diameter, delineated by syntaxin, SNAP 25 and N-type calcium channels and that synaptic vesicles are concentrated at these sites as indicated by the localisation of VAMP. Most of these terminal varicosities possess active zones that are precisely apposed to large clusters of P2X(1) receptors. However small clusters of P2X(2) to P2X(6) receptors can be found that are near the strings of varicosities but not usually coincidental with them except P2X(3). The functional significance of this arrangement of vesicle-associated proteins and P2X receptors for the generation of synaptic potentials at the autonomic neuromuscular junction is discussed.