Deconstructing complexity: serial block-face electron microscopic analysis of the hippocampal mossy fiber synapse - PubMed (original) (raw)

Deconstructing complexity: serial block-face electron microscopic analysis of the hippocampal mossy fiber synapse

Scott A Wilke et al. J Neurosci. 2013.

Abstract

The hippocampal mossy fiber (MF) terminal is among the largest and most complex synaptic structures in the brain. Our understanding of the development of this morphologically elaborate structure has been limited because of the inability of standard electron microscopy techniques to quickly and accurately reconstruct large volumes of neuropil. Here we use serial block-face electron microscopy (SBEM) to surmount these limitations and investigate the establishment of MF connectivity during mouse postnatal development. Based on volume reconstructions, we find that MF axons initially form bouton-like specializations directly onto dendritic shafts, that dendritic protrusions primarily arise independently of bouton contact sites, and that a dramatic increase in presynaptic and postsynaptic complexity follows the association of MF boutons with CA3 dendritic protrusions. We also identify a transient period of MF bouton filopodial exploration, followed by refinement of sites of synaptic connectivity. These observations enhance our understanding of the development of this highly specialized synapse and illustrate the power of SBEM to resolve details of developing microcircuits at a level not easily attainable with conventional approaches.

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Figures

Figure 1.

Figure 1.

Application of SBEM to the study of hippocampal MF terminals. A, Schematic representation of the hippocampus with the region of SBEM data collection (CA3 SL) indicated (dashed box). B, DiI-labeled MFBs. Scale bar, 5 μm. C, CA3 neuron with TE spines labeled by intracellular injection of LY dye in fixed tissue. Scale bar, 5 μm. D, Reconstruction of CA3 neuron apical dendritic shaft (red), with TE spines each in a separate color. Scale bar, 2 μm. Numbered lines demonstrate planes of SBEM data, which correspond to raw data shown in E1–E4. E1, Full field of view image from SBEM instrument; dashed box is region of interest for subsequent sections in E2–E4. N, Cell nucleus. E2–E4, Raw data images corresponding to planes as indicated in D with contours pseudocolored in correspondence with reconstruction in D. Scale bar, 1 μm.

Figure 2.

Figure 2.

Reconstruction of large volumes of CA3 SL with high resolution at the ultrastructural level. A, Representative sample of reconstructed CA3 SL neuropil at P14, showing one plane of raw SBEM data. Data are ∼25 μm in the x and y dimension and ∼20 μm in the z dimension. Scale bar, 2 μm. A number of dendritic shafts are visible along with reconstructed synaptic components. B, One CA3 apical shaft reconstruction from A, with a single MFB (shaft, pink; spines, red; bouton, blue. Scale bar, 2 μm. C, A portion of the reconstructed dataset shown in B, rendered emerging from a single plane of raw data. Scale bar, 2 μm. D, Non-rendered version of the data plane shown in C, with object contours shown in their corresponding colors (shaft, pink; spines, red; bouton, blue; other boutons not shown in C indicated in green; neighboring shafts, asterisks). Scale bar, 2 μm. E, High-magnification image demonstrating SBEM resolution of ultrastructural features at P14. Arrowheads (red) indicate sites of synaptic release with PSDs apposed to presynaptically clustered vesicles in an MF terminal. Single presynaptic vesicles can be resolved (yellow arrowheads), as well as various organelles (mitochondria, blue arrowheads). F, Presynaptic and postsynaptic structures from E are shaded for easier identification: postsynaptic shaft and spine heads (Dend/TE), red; MFB, yellow; PSDs, green; mitochondria (Mito), blue. Scale bars: E, F, 500 nm.

Figure 3.

Figure 3.

Synapse initiation and development of bouton ultrastructure. A, P0: early vesicle clustering at site of axonal contact with CA3 dendrite. B, Micrograph from A without pseudocoloring. C, P7: early MFB contacting CA3 dendrite in the absence of immature spine. D, Micrograph from C without pseudocoloring. E, P7: early association of axonal and dendritic specializations. F, P7: early dendritic protrusion emerging in contact with glial processes. G, P0 bouton with reconstructed ultrastructure. H, P7 bouton with reconstructed ultrastructure. I, P14 bouton with reconstructed ultrastructure. J, Adult bouton with reconstructed ultrastructure. K, Magnified view of vesicle density from I. L, Magnified view of vesicle density from J. Structure color code: axon, red; bouton, blue; vesicles, orange; mitochondria, white; TE spines and spine precursors, green; glial processes, yellow; dendrite shaft, orange. Arrowheads in A and C indicate mitochondrial profiles. Scale bars: 1 μm; insets K and L, 10 nm.

Figure 4.

Figure 4.

Maturation of MF axonal specializations. MF axons (red) with boutons (regions of vesicle accumulation; blue) were reconstructed from SL samples. A–D, Representative MF terminals and axons from P0 (A), P7 (B), P14 (C), and adult (D) mice. E, Quantification of the mean volume of the vesicle-containing portion of MFBs excluding volume of filopodial protrusions for each age. F, Quantification of the mean number of filopodial extensions per bouton for each age. Scale bars, 2 μm. *p < 0.05, **p < 0.01, ***p < .001 by ANOVA. Error bars represent ±SEM.

Figure 5.

Figure 5.

Analysis of the maturation of CA3 dendrite TE structure. Postsynaptic protrusions with a common neck were reconstructed as a single object for analysis. A–D, Representative TE spines from P0 (A), P7 (B), P14 (C), and adult (D) mice. E–H, TE spine volumes were quantified for each age. Histograms of binned spine volumes from P0 (E), P7 (F), P14 (G), and adult (H) mice are displayed. Scale bars, 2 μm.

Figure 6.

Figure 6.

Structural relationships between axonal and dendritic elements demonstrated by reconstruction of large segments of CA3 dendrite with inputs. A–D, Reconstruction of dendritic structure in which dendritic shafts are indicated in silver, and each dendritic protrusion is indicated in a separate color for P0, P7, P14, and adult (P120). E–H, Same reconstructions shown in A–D, with dendritic protrusions indicated in red. I–L, Same dendritic reconstructions shown in E–H but with a subset of the reconstructed axons and MFBs that form contacts onto that neuron, each shown in a separate color. M, Quantification of mean number of TE spines contacted per MFB for each age. N, Plots of the association between the volume of individual MFBs and the number of TE spines, which they contact at each age. O, Quantification of the percentage of MFBs at each age, which are associated with a dendritic protrusion, and the percentage of TE spines, which are associated with a MFB at each age. Scale bar, 2 μm. ***p < 0.001, by t test.

Figure 7.

Figure 7.

Glial cells contact sites of initial spine outgrowth. A, Dendritic shaft (silver) and spines (red) shown at P7 in close association with three separately reconstructed glial cell processes (blue, green, and pink). B, P14 dendritic shaft (silver) and spines (red) shown in close association with a single large, partially reconstructed glial cell (yellow). C–F, EM cross-sections showing glia (green), dendritic shafts (orange), dendritic protrusions (red), and axonal structures (blue). C, P7: immature dendritic protrusion forming initial contact with undifferentiated axon, in close association with glial processes. D, P7: immature dendritic protrusion only in contact with glial processes. E, P14: glial processes in association with synaptic terminals. F, Adult: glial processes surrounding synaptic terminals. G, Enlarged micrograph from E without pseudocoloring. H, Enlarged micrograph from F without pseudocoloring. Scale bars: A, B, 2 μm; C–H, 1 μm.

Figure 8.

Figure 8.

Single TE spines can receive synaptic contacts from multiple MFBs. A, Two reconstructed P14 MFBs (orange and green) shown in contact with a CA3 dendritic shaft (violet). For simplicity, only a single relevant TE spine is shown (purple), essentially obscured in A by the boutons. B, Individual reconstructed elements from A shown separately. Single TE spine is clearly visible here (purple). C, Single cross-section of ultrastructural data from reconstruction with colors as indicated in A. PSDs (red) at the interface between purple spine and each bouton are clearly visible and indicated with a red arrowhead. D, Same ultrastructural cross-section as E, without shading for a clearer view of ultrastructure. Scale bars, 2 μm.

Figure 9.

Figure 9.

Single MFBs can contact TE spines on dendrites from two separate neurons. A, Reconstructions of two separate P14 dendrites (olive with green spines and pink with red spines) are shown in contact with a single reconstructed presynaptic bouton (blue). Only a subset of relevant spines is shown for ease of interpretation. B, Higher-magnification view of dashed box in A. C, Each individual element as in A, shown separately. D, Single cross-section of ultrastructural data used for this reconstruction, showing bouton (blue) and spines (green and red). PSDs (yellow) are clearly visible between the bouton and spines from each neuron as indicated by red arrowheads. E, Same cross-section as in E, without shading for a clearer view of ultrastructure. Scale bars, 2 μm.

Figure 10.

Figure 10.

Refinement of MF–CA3 synaptic contacts during postnatal development. A–C, Example TE spines at P7, P14, and adult time points (red) with synaptic sites (blue) (inset represents each synaptic site viewed head on), the number of synaptic contacts per spine is represented in terms of relative frequency as a wedge diagram for each developmental stage. D, Quantification of the number of synaptic sites relative to TE volume in cubic micrometers for P7, P14, and adult time points. E, Cumulative distribution plot of synaptic site surface areas in square micrometers for P14 and adult time points. F, Quantification of the percentage of TE surface area occupied by synaptic sites for P14 and adult time points. G, Scatter plot representation of the relationship between TE surface area and number of synaptic sites for P7, P14, and adult time points. Scale bar, 1 μm. *p < 0.05, ***p < 0.001, by t test. Error bars represent ±SEM.

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