Spatial relationships between neuromuscular junctions and microvessels in hamster cremaster muscle

Acetylcholine (ACh) dilates arterioles in skeletal muscle. Neuromuscular junctions (NMJs) are the known source of ACh in this tissue. We hypothesized that microvascular topology is related to the distribution of NMJs. To test this, the spatial relationships between NMJs, arterioles, and capillaries in the hamster cremaster muscle were investigated. Male hamsters (n = 5, 80-100 g) were anesthetized (sodium pentobarbital, 60 mg/kg) and the cremaster was perfused with fluorescein isothiocyanate-bovine serum albumin (FITC-BSA) and Microfil compound. Excised muscles were stained for NMJs (cholinesterase reaction) and cleared in glycerin. Grid overlays divided each cremaster evenly into proximal and distal regions and 40 numbered 3 × 3-mm study fields. Five fields/region (˜25% of muscle area) were chosen randomly. Point counting ('counts') on a coherent test grid (component grid dimensions at x 144 magnification: 150 × 150, 300 × 300, and 450 × 450 μm) quantified NMJs, arterioles, and capillaries, respectively. NMJ:arteriole and NMJ:capillary nearest distances were obtained and arterioles nearest NMJs were classified by branch order. Filling with FITC-BSA vs Microfil indicated that all arterioles and ˜92% of capillaries were perfused with Microfil. Relative counts (i.e., volume fractions) for capillaries were five- to sixfold greater than those for arterioles, which were two- to fivefold greater than those for NMJs. Capillary counts were similar between muscle regions and did not correlate with NMJ counts. In the distal muscle, arteriole and NMJ counts were correlated (r = 0.55, P < 0.05) and counts for both structures were greater than those in proximal regions. NMJ:capillary distances (proximal, 11.9 ± 0.9 μm; distal, 14.5 ± 0.6 μm) were less (P < 0.05) than respective NMJ:arteriole distances (111.1 ± 7.1 and 89.8 ± 3.2 μm). Fourth- and fifth-order arterioles accounted for 69% of arterioles nearest NMJs. These findings suggest that NMJs may provide a vasoactive stimulus which varies with muscle region and with location in the microvascular network.