High-Resolution Synaptic Connectomics

Abstract

high-speed, high-resolution connectomics enables unambiguous mapping of synapses, gap junctions, adherens junctions, and other forms of adjacency among neurons in complex neural systems such as brain and retina. This chapter reviews the motivations for generating complete network architectures; the technologies available for large-scale network acquisition, visualization, and analysis; the fusion of molecular markers with a high-resolution ultrastructure; new networks and organelles discovered by ultrastructural connectomics; and new technological advances needed to expand the applications of connectomics. Motivations for Ultrastructural Connectomicsa connectome is the complete set of cellular partners and connections for a neural region. it can be executed on the mesoscale (spatial resolution of magnetic resonance imaging or even conventional optical imaging) to map fiber networks or on the nanoscale (spatial resolution of electron imaging) to map synaptic networks. This review addresses our experience with high-resolution synaptic connectomics based on automated transmission electron microscope (aTeM) imaging.The notion of using computational methods to accelerate ultrastructural analysis is at least three decades old [92]. even so, computational imaging for electron microscopy did not become a mainstream strategy until recently. There were three reasons for this: slow acquisition speed, expensive storage, and weak analytical scale. Film-based imaging followed by high-performance digitization [40] or even digital camera acquisition followed by analysis was so slow that it had no competitive advantage. Second, data storage at the resolution required for synaptic identification and quantification was prohibitively expensive, especially for Nih-funded investigators. The third reason is less obvious: no formal rationale existed to motivate large-scale acquisitions.

Rod‐cone crossover connectome of mammalian bipolar cells

“…We have not discovered any BC input to these bistratified γACs. Since our connectomics resolution (2 nm) in the projection TEM images of volume ensures that we do not miss any synapses (Marc et al, ) and since γAC 5281 is largely contained within the RC1 volume, it appears that it truly lacks any BC input. Motif C3 bistratified γACs are then presynaptic to ON cone BCs (Fig.…”

“…Arrows indicate dendro/axosomatic synapses. G: A composite of three sections to visualize four dendro/axosomatic synapses (11651,11683,43130,43114) volume ensures that we do not miss any synapses (Marc et al, 2014b) and since gAC 5281 is largely contained within the RC1 volume, it appears that it truly lacks any BC input. Motif C3 bistratified gACs are then presynaptic to ON cone BCs (Fig.…”

“…Our connectome methods, tissue sampling, and analytical techniques have been described previously in detail (Anderson et al, ; Lauritzen et al, ; Marc et al, ) and only essential points are expanded here. Connectome volume RC1 is an open‐access, transmission electron microscopy (TEM)‐based, ultrastructural rabbit retina volume based on 371 serial 70–90‐nm‐thick sections, capstoned and intercalated with optical sections containing small molecule signals for cell classification (Anderson et al, ).…”

“…Using molecular markers (Marc et al, 1995;Kalloniatis et al, 1996) and excitation mapping with 1amino-4-guanidobutane (Marc, 1999a,b;Marc and Jones, 2002), every neuron with a soma in RC1 can be classified using isodata clustering (Marc and Jones, 2002) and/or principal components mapping (Anderson et al, 2009) essentially without error as a horizontal IgG B100R AB_2532053 Signature Immunologics, Torrey, UT 1:100 anti-L-aspartate IgG D100R AB_2341093 Signature Immunologics, Torrey, UT 1:100 anti-L-glutamate IgG E100R AB_2532055 Signature Immunologics, Torrey, UT 1:100 anti-glycine IgG G100R AB_2532057 Signature Immunologics, Torrey, UT 1:100 anti-L-glutamine IgG Q100R AB_2532059 Signature Immunologics, Torrey, UT 1:100 anti-taurine IgG TT100R AB_2532060 Signature Immunologics, Torrey, UT 1:100 anti-GABA IgG YY100R AB_2532061 Signature Immunologics, Torrey, UT 1:100 cell, OFF cone BC, ON cone BC, rod BC, narrow-field GAC, wide-field gAC, GC, or M€ uller cell (Anderson et al, 2009(Anderson et al, , 2011b. Many crossing processes in RC1 originating from GCs and ACs outside the volume traverse one or more intercalated GABA, glycine, 1-amino-4-guanidobutane, or taurine channels, which often allows direct biochemical classification (Marc et al, 2013(Marc et al, , 2014a(Marc et al, , 2014b. More important, each molecular classifier uniquely maps onto the distinctive morphologies of each class of cells in 3D (including fine-scale stratification in the inner plexiform layer) and unique collections of network motifs accessed by each.…”

Heterocellular Coupling Between Amacrine Cells and Ganglion Cells

“…At high resolution, it is clear that most gap junctions are not fasciculations but rather crossings (Figure 10 Column 1). The resolution of RC1 (2.18 nm/pixel) is sufficient to reliably detect gap junctions and measure their areas (Figure 10 Column 2) but is not adequate for complete validation as occasional adherens junctions can mimic gap junctions in oblique view ( Marc et al, 2014b ). High resolution (0.27 nm/pixel) reimaging with goniometric tilt allows validation of gap junctions by visualizing their characteristic multilaminar density profiles (Figure 10 Columns 3, 4 and inset).…”