Tag Archives: Crystal L. Sigulinsky

Ultrastructural Connectomics Reveals The Entire Chemical And Electrical Synaptic Cohort Of An ON Cone Bipolar Cell In The Inner Plexiform Layer Of The Rabbit Retina

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This abstract was presented at the 2014 Society for Neuroscience meeting in Washington D.C. by J. Scott Lauritzen, Crystal L. Sigulinsky, Danny P. Emrich, Joshua M. Dudleston, Noah T. Nelson, Rebecca L. Pfeiffer, Nathan R. Sherbotie, John V. Hoang, Jefferson R. Brown, Carl B. WattJames R. Anderson, Bryan W. Jones and Robert E. Marc.

Purpose: Despite large-scale efforts aimed at mapping the mammalian nervous system, the entire synaptic cohort of a single mammalian neuron of any class has never been mapped. To this end we reconstructed all chemical and electrical synaptic partners of a single ON cone bipolar cell (ON CBC) in the inner plexiform layer (IPL) of the rabbit retina. We then searched all members of the same cell class for repeating network motifs and explored postsynaptic cell sampling topologies from this bipolar cell (BC).

Methods: Cells in retinal connectome 1 (RC1) were annotated with Viking viewer, and explored via graph visualization of connectivity and 3D rendering (Anderson et al., 2011 J Microscopy). Small molecule signals in RC1, e.g. GABA, glycine, and L-glutamate, combined with morphological reconstruction and connectivity analysis allow robust cell classification. The default resolution of RC1 is 2.18nm/pixel, however goniometric recapture at 0.273 nm/pixel was performed as needed for synapse verification.

Results: ON CBC 593 is one of 20 BCs of this class in RC1, the axonal arbors of which tile with gap junctions between nearest neighbors at their distal axonal tips. ON CBC 593 contains 194 ribbons, 274 postsynaptic densities, 20 gap junctions, and 66 conventional synapses, for a total of 554 synaptic connections. Twenty ganglion cells sample the glutamatergic output. ON CBC 593 is presynaptic to 262 amacrine cell (AC) processes, and is postsynaptic to 228 AC processes. Of these, 33% form reciprocal connections. We approximate that ON CBC 593 forms synapses with 50 distinct ACs. ON CBC 593 is routinely pre- and postsynaptic to within-class, cross-class, feedback, and feedforward inhibition motifs, including 1 instance of OFF-ON crossover inhibition. ON CBC 593 forms 12 gap junctions with at least 2 AII ACs, 7 with 5 ON CBCs, and 1 with itself. We searched for repeating network motifs across all ON CBCs of this class in RC1. Thus far, 80% of these form in-class inhibitory motifs, and 75% form cross-class inhibitory motifs. All ACs and GCs discovered to contact multiple branches of ON CBC 593 form synapses on every branch.

Conclusions: An individual bipolar cell is inherently multi-kinetic, receiving inhibition driven by all ON CBC classes, sharing these signals via gap junctions with ON CBCs of the same class, and driving inhibition of all ON CBC classes. This constitutes a substrate for multi-channel coordination throughout the IPL, and predicts multi-kinetic BC responses. The results establish a normative framework against which members of the same and different classes may be compared, and foster interpretation of BC physiological behavior under different stimulus regimes.

Ultrastructural Reconstruction of ON Cone Bipolar Cell Projective Fields In The Innter Plexiform Layer of The Rabbit Retina

retina reconstruction

This abstract was presented at the 2014 FASEB Summer Research Conference in Saxtons River, Vermont by J. Scott Lauritzen, Crystal L. Sigulinsky, Noah T. Nelson, Nathan R. Sherbotie, Danny P. Emrich, Rebecca L. Pfeiffer, Jefferson R. Brown, John V. Hoang, Joshua M. Dudleston, Carl B. Watt, Kevin Rapp, Marguerite V. Shaw, Jia-Hui Yang, James R. Anderson, Bryan W. Jones and Robert E. Marc.

Purpose: Functional mapping in tiger salamander shows that bipolar cell (BC) projective fields far exceed their axonal fields, and directly implicates wide-field GABAergic amacrine cells (wf γACs) and gap junctions (Asari & Meister, 2014). Strikingly, single BCs exert differential effects on functionally distinct ganglion cells (GCs), likely achieved by privatized amacrine cell (AC) presynaptic inhibition to specific BC-GC synaptic pairs (Asari & Meister, 2012). To address whether BC projective fields in the mammal are equally broad, wf γAC- and gap junction-dependent, and GC type unselective, we reconstructed all electrical and chemical synaptic partners of a single ON cone BC in the inner plexiform layer of the rabbit retina, and searched BC-GC synaptic pairs for differential synaptic inhibition.

Methods: Cells in retinal connectome 1 (RC1) were annotated with Viking viewer, and explored via connectivity visualizations and 3D rendering (Anderson et al., 2011). Small molecule signals embedded in RC1, e.g. GABA, glycine, and L-glutamate, combined with morphological reconstruction and connectivity analysis allow robust cell classification. We used the MacNeil et al. (2004) rabbit BC classification scheme.

Results: CBb5w 593 is one of 20 ON cone BCs of this class in RC1. This CBb5w is presynaptic to 17 distinct GCs and 262 AC processes, and postsynaptic to 228 AC processes. The majority of these ACs are wf γACs. We estimate this BC forms synapses with 50 unique ACs. Asari & Meister (2014) found that single bipolar cell projective fields range up to 1 mm, far beyond a BC axonal field, and differentially drive multiple classes of GC. We discovered BC-BC within- and cross-class coupling and lateral inhibition that construct sign-conserving and sign-inverting projective fields to many distinct ganglion cell classes across the entire 0.25 mm diameter of RC1, much greater than a 60 µm BC axonal field. Cross-class projections access a broader set of GCs than expected from in-class projections alone. The BC-BC coupling is independent of BC-AII AC coupling. 94% of the CBb5w 593 BC-GC synaptic pairs receive feedback inhibition within the varicosity of the ribbon, but the number of feedback synapses is highly variable (coefficient of variation = 0.81). 35% of the BC-GC pairs receive feedforward inhibition within 2 microns of the postsynaptic density.

Conclusions: Mammalian BCs use novel cross-class topologies to distribute signals to a wide range of GCs and establish projective fields similar to those discovered in non-mammalian species. BC-BC within- and cross-class coupling and lateral inhibition via wf γACs establish sign-conserving and sign-inverting projective fields, respectively, up to 1 mm diameters. BC-GC synaptic pairs overwhelmingly employ feedback vs. feedforward inhibition to modulate signaling, and the numbers of feedback synapses are highly variable across these pairs, accounting for privatized and differential GC responses to the same BC drive.

A Synaptic Basis for Small World Network Design in the ON Inner Plexiform Layer of the Rabbit Retina

Bipolar cells_

This abstract was presented today at the 2014 Association for Research in Vision and Opthalmology (ARVO) meetings in Orlando, Florida by J Scott Lauritzen, Noah T. Nelson, Crystal L. Sigulinsky, Nathan Sherbotie, John Hoang, Rebecca L. PfeifferJames R. Anderson, Carl B. Watt, Bryan W. Jones and Robert E. Marc.

Purpose: Converging evidence suggests that large- and intermediate-scale neural networks throughout the nervous system exhibit small world’ design characterized by high local clustering of connections yet short path length between neuronal modules (Watts & Strogatz 1998 Nature; Sporns et al.2004 Trends in Cog Sci). It is suspected that this organizing principle scales to local networks (Ganmor et al. 2011 J Neurosci; Sporns 2006 BioSystems) but direct observation of synapses and local network topologies mediating small world design has not been achieved in any neuronal tissue. We sought direct evidence for synaptic and topological substrates that instantiate small world network architectures in the ON inner plexiform layer (IPL) of the rabbit retina. To test this we mined ≈ 200 ON cone bipolar cells (BCs) and ≈ 500 inhibitory amacrine cell (AC) processes in the ultrastructural rabbit retinal connectome (RC1).

Methods: BC networks in RC1 were annotated with the Viking viewer and explored via graph visualization of connectivity and 3D rendering (Anderson et al. 2011 J Microscopy). Small molecule signals embedded in RC1 e.g. GABA glycine and L-glutamate combined with morphological reconstruction and connectivity analysis allow for robust cell classification. MacNeil et al. (2004 J Comp Neurol) BC classification scheme used for clarity.

Results: Homocellular BC coupling (CBb3::CBb3 CBb4::CBb4 CBb5::CBb5) and within-class BC inhibitory networks (CBb3 → AC –| CBb3 CBb4 → AC –| CBb4 CBb5 → AC –| CBb5) in each ON IPL strata form laminar-specific functional sheets with high clustering coefficients. Heterocellular BC coupling (CBb3::CBb4 CBb4::CBb5 CBb3::CBb5) and cross-class BC inhibitory networks (CBb3 → AC –| CBb4 CBb4 → AC –| CBb3 CBb4 → AC –| CBb5 CBb5 → AC –| CBb4 CBb3 → AC –| CBb5 CBb5 → AC –| CBb3) establish short synaptic path lengths across all ON IPL laminae.

Conclusions: The retina contains a greater than expected number of synaptic hubs that multiplex parallel channels presynaptic to ganglion cells. The results validate a synaptic basis (ie. direct synaptic connectivity) and local network topology for the small world architecture indicated at larger scales providing neuroanatomical plausibility of this organization for local networks and are consistent with small world design as a fundamental organizing principle of neural networks on multiple spatial scales.

Support:  NIH EY02576 (RM), NIH EY015128 (RM), NSF 0941717 (RM), NIH EY014800 Vision Core (RM), RPB CDA (BWJ), Thome AMD Grant (BWJ).