Fig. 3. AChR-ß^3F/3F mice fail to form morphologically complex synapses. (A-H) Confocal images of synapses, stained with Alexa Fluor 594--BGT, in the diaphragm muscle from P30 wild-type and AChR- ß^3F/3F mice. E-H are higher magnification images of the boxed regions in A-D. Scale bar: 10 µm for A-D and 5 µm for E-H. (I) At category 1 synapses, >75% of the surface area is covered with AChR striations; at category 2 synapses, 25-75% of the surface area is covered with AChR striations; at category 3 synapses, <25% of the surface area is covered with AChR striations. AChR-ß^3F/3F mice have fewer category 1 synapses and more category 3 synapses than wild-type mice. Wild type: 39 category 1 synapses, 20 category 2 synapses, 0 category 3 synapses; AChR-ß^3F/3F: 1 category 1 synapse, 18 category 2 synapses, 36 category 3 synapses; P<0.005, ² test. (J) Two or fewer gaps are evident at category 1 synapses; three or four gaps are evident at category 2 synapses; five or more gaps are evident at category 3 synapses. AChR- ß^3F/3F mice have fewer category 1 synapses and more category 3 synapses than wild-type mice. Wild type: 40 category 1 synapses, 16 category 2 synapses, 3 category 3 synapses; AChR-ß^3F/3F: 11 category 1 synapses, 26 category 2 synapses, 18 category 3 synapses; P<0.005, ² test. (K-N) Ultrastructural analysis of wild-type and AChR-ß^3F/3F mice. (K,L) Presynaptic nerve terminals in wild-type (K) and AChR-ß^3F/3F (L) mice are filled with synaptic vesicles and are apposed by postjunctional folds. Scale bar: 500 nm. (M,N) The postsynaptic membrane in wild-type (M) and AChR-ß^3F/3F (N) mice is electron dense at the tops of the postjunctional folds (arrows). Scale bar=500 nm. (O) The number of total postjunctional folds and the fold index are normal in AChR-ß^3F/3F mice, but the number of openings, or mouths, to the synaptic cleft is reduced in AChR-ß^3F/3F mice. Number of total postjunctional folds: wild type, 0.69±0.04 folds/µm, n=57; AChR-ß^3F/3F, 0.70±0.03 folds/µm, n=75; P>0.05, unpaired t-test. The fold index: wild type, 4.5±0.2, n=57; AChR-ß^3F/3F, 4.2±0.2%, n=75; P>0.05, unpaired t-test. The number of postjunctional fold mouths: wild type, 0.38±0.02 mouths/µm, n=57; AchR-ß^3F/3F, 0.24±0.01 mouths/µm, n=75; P<0.005. (P,Q) A model to illustrate the organization of postjunctional folds in wild-type (P) and AChR-ß^3F/3F (Q) mice. In AChR-ß^3F/3F mice, the number of postjunctional folds, lined with basal lamina, is normal, but fewer postjunctional folds have mouths that open to the synaptic cleft; as a consequence, the number of AChR striations is reduced in AChR- ß^3F/3F mice (E,F versus G,H).