PurposeA connectome is a comprehensive description of synaptic connectivity for a neural domain. Our goal was to produce a connectome data set for the inner plexiform layer of the mammalian retina. This paper describes our first retinal connectome, validates the method, and provides key initial findings.MethodsWe acquired and assembled a 16.5 terabyte connectome data set RC1 for the rabbit retina at ≈2 nm resolution using automated transmission electron microscope imaging, automated mosaicking, and automated volume registration. RC1 represents a column of tissue 0.25 mm in diameter, spanning the inner nuclear, inner plexiform, and ganglion cell layers. To enhance ultrastructural tracing, we included molecular markers for 4-aminobutyrate (GABA), glutamate, glycine, taurine, glutamine, and the in vivo activity marker, 1-amino-4-guanidobutane. This enabled us to distinguish GABAergic and glycinergic amacrine cells; to identify ON bipolar cells coupled to glycinergic cells; and to discriminate different kinds of bipolar, amacrine, and ganglion cells based on their molecular signatures and activity. The data set was explored and annotated with Viking, our multiuser navigation tool. Annotations were exported to additional applications to render cells, visualize network graphs, and query the database.ResultsExploration of RC1 showed that the 2 nm resolution readily recapitulated well known connections and revealed several new features of retinal organization: (1) The well known AII amacrine cell pathway displayed more complexity than previously reported, with no less than 17 distinct signaling modes, including ribbon synapse inputs from OFF bipolar cells, wide-field ON cone bipolar cells and rod bipolar cells, and extensive input from cone-pathway amacrine cells. (2) The axons of most cone bipolar cells formed a distinct signal integration compartment, with ON cone bipolar cell axonal synapses targeting diverse cell types. Both ON and OFF bipolar cells receive axonal veto synapses. (3) Chains of conventional synapses were very common, with intercalated glycinergic-GABAergic chains and very long chains associated with starburst amacrine cells. Glycinergic amacrine cells clearly play a major role in ON-OFF crossover inhibition. (4) Molecular and excitation mapping clearly segregates ultrastructurally defined bipolar cell groups into different response clusters. (5) Finally, low-resolution electron or optical imaging cannot reliably map synaptic connections by process geometry, as adjacency without synaptic contact is abundant in the retina. Only direct visualization of synapses and gap junctions suffices.ConclusionsConnectome assembly and analysis using conventional transmission electron microscopy is now practical for network discovery. Our surveys of volume RC1 demonstrate that previously studied systems such as the AII amacrine cell network involve more network motifs than previously known. The AII network, primarily considered a scotopic pathway, clearly derives ribbon synapse input from photopic ON and OFF cone bipol...

Disruption of endothelial barrier is a critical pathophysiological factor in inflammation. Thrombin exerts a variety of cellular effects including inflammation and apoptosis through activation of the protease activated receptors (PARs). The activation of PAR-1 by thrombin is known to have a bimodal effect in endothelial cell permeability with a low concentration (pM levels) eliciting a barrier protective and a high concentration (nM levels) eliciting a barrier disruptive response. It is not known whether this PAR-1-dependent activity of thrombin is a unique phenomenon specific for the in vitro assay or it is part of a general anti-inflammatory effect of low concentrations of thrombin that may have a physiological relevance. Here, we report that low concentrations of thrombin or of PAR-1 agonist peptide induced significant anti-inflammatory activities. However, relatively high concentration of thrombin or of PAR-1 agonist peptide showed pro-inflammatory activities. By using function-blocking anti-PAR-1 antibodies and PI3 kinase inhibitor, we show that the direct antiinflammatory effects of low concentrations of thrombin are dependent on the activation of PAR-1 and PI3 kinase. These results suggest a role for cross communication between PAR-1 activation and PI3 kinase pathway in mediating the cytoprotective effects of low concentrations of thrombin in the cytokine-stimulated endothelial cells.

No abstract

Atopic dermatitis (AD), also known as atopic eczema, is chronic pruritic skin disease. AD can increase psychological stress as well, increasing glucocorticoid release and exacerbating the associated symptoms. Chronic glucocorticoid elevation disturbs neuroendocrine signaling and can induce neuroinflammation, neurotoxicity, and cognitive impairment; however, it is unclear whether AD-related psychological stress elevates glucocorticoids enough to cause neuronal damage. Therefore, we assessed the effects of AD-induced stress in a mouse AD model. AD-related psychological stress increased astroglial and microglial activation, neuroinflammatory cytokine expression, and markers of neuronal loss. Notably, melatonin administration inhibited the development of skin lesions, scratching behavior, and serum IgE levels in the model mice, and additionally caused a significant reduction in corticotropin-releasing hormone responsiveness, and a significant reduction in neuronal damage. Finally, we produced similar results in a corticosterone-induced AD-like skin model. This is the first study to demonstrate that AD-related psychological stress increases neuroendocrine dysfunction, exacerbates neuroinflammation, and potentially accelerates other neurodegenerative disease states.

No abstract