Examining Hippocampal Mossy Fiber Synapses by 3D Electron Microscopy in Wildtype and Kirrel3 Knockout Mice

Reconstructing MF presynaptic complexes by SBEM. A, Diagram of hippocampal MF circuitry. Boxed region notes the tissue area analyzed. On the right, the dark blue outline identifies the DG neuron. B, Representative image of a wildtype SBEM section showing MF synapse components: main MF bouton (dark blue), MF filopodia (light blue), DG axon (yellow), TEs (orange), SVs (red arrows), and mitochondria (white arrows). Scale bar, 1 μm. C, 3D reconstruction of the MF presynaptic complex in B. The postsynaptic TEs are not shown. Dotted line in C shows the location of slice shown in B. D,E, Sample wildtype (D) and Kirrel3 knockout (E) 3D reconstructions showing eight MF presynaptic complexes each.

MF filopodia exist in three states during development. A, The average volume of the main MF bouton per genotype. p = 0.5102. B, The average number of MF filopodia per main MF bouton. p = 0.1323. C, Cumulative histogram showing the number of MF filopodia per main MF bouton in each genotype. Note: Kirrel3 knockouts tend to have MF boutons with fewer filopodia. p = 0.2360. D–F, Representative images of MF filopodia in each of the three synaptic states (synapse-free, partial, complete). Yellow arrow indicates PSD. For all graphs: sample size: WT = 30 and KO = 34 main MF boutons. Error bars show mean ± SEM. Scale bars, 1 μm.

Kirrel3 regulates MF filopodial synapse density. A, Table showing percentage of MF filopodia that are synapse-free, or contain partial or complete synapses. Note: An individual MF filopodia can have multiple synapses, thus the totals do not sum to 100%. B–D, Quantification of the number of synapse-free MF filopodia (B), partial synapses (C), and complete synapses (D) per main MF bouton by genotype. E, Quantification of total number of synapses (partial + complete) per main MF bouton. *p = 0.0357 using a two-tailed t test. Sample size for A–E: WT = 30 and KO = 34 main MF boutons. F, Quantification of the number of SVs in complete synapses. Sample size for F: WT = 55 and KO = 42 synapses. G, Quantification of the number of SVs in partial synapses. Sample size for G: WT = 150 and KO = 131 synapses. For all graphs: Unless noted, p > 0.05 (Table 1). Error bars show mean ± SEM.

Kirrel3 regulates MF filopodial synapses with GABA neurons, but not CA3 neurons. A,B, 2D (A) and 3D (B) example of a GABA dendrite (green) with simple spine-like protrusions and no PSDs with main MF boutons. Dotted line in B shows the location of slice shown in A. C,D, 2D (C) and 3D (D) example of a CA3 neuron (orange) with multiheaded TE spines and many PSDs with main MF boutons. Dotted line in D shows the location of slice shown in C. E,F, Representative images of complete MF filopodial synapses made onto a GABA dendritic shaft (E) and a GABA spine (F). MF filopodia (blue), GABA dendrite (green), synapses indicated by white arrows. G,H, Representative images of complete MF filopodial synapses made onto a second CA3 dendritic shaft (G) and a CA3 TE (H). Main MF bouton and MF filopodia (blue), CA3 dendrite and TEs (orange), second CA3 dendrite (yellow), synapses indicated by white arrows. I–L, Quantification of MF filopodial synapses onto different types of postsynaptic neurons. I, Percentage of wildtype or Kirrel3 knockout MF filopodia making complete synapses with indicated postsynaptic partner type. J, Number of complete MF filopodial synapses onto GABA neurons per main MF bouton. **p = 0.0075 by Mann-Whitney test. K, Number of complete MF filopodial synapses onto CA3 neurons per main MF bouton. L, Including only the MF filopodia that make a complete synapse, the percentage of each partner type is shown. Sample size for I–L: WT = 30 and KO = 34 main MF boutons. Unless indicated p > 0.05. M–P, Comparison of the morphology of filopodia synapsing with GABA versus CA3 neurons in wildtype and Kirrel3 knockout mice using the Kruskal-Wallis test with multiple comparisons. M, Volume of filopodia in μm³. GABA WT to KO *p = 0.0419, CA3 WT to KO *p = 0.0507. N, Length of filopodia in micrometers. *p = 0.0299. O, Cross sectional area in square micrometers. p > 0.05. P, Number of SVs per synapse. p > 0.05. Sample size for M–O: WT GABA = 20, KO GABA = 4, WT CA3 = 27, KO CA3 = 31. Sample size for P: WT GABA = 20, KO GABA = 5, WT CA3 = 27, KO CA3 = 33. For entire figure, error bars show mean ± SEM. Scale bars, 1 μm.

Kirrel3 does not regulate en passant synapse density. A–C, 3D example (A) of a spiny GABA dendrite targeted by en passant synapses from two separate DG axons. Synapses magnified in B, C. GABA dendrite (green), DG axon (yellow), en passant synapse (pink). D, Percentage of en passant synapses made onto GABA and CA3 dendrites from 73 WT and 68 KO DG axon segments. E, Table reporting the number of en passant synapses per millimeter axon. Scale bars, 1 μm.