Mapping Synaptic Input Fields of Neurons with Super-Resolution Imaging

Abstract

Summary As a basic functional unit in neural circuits, each neuron integrates input signals from hundreds to thousands of synapses. Knowledge of the synaptic input fields of individual neurons, including the identity, strength and location of each synapse, is essential for understanding how neurons compute. Here we developed a volumetric super-resolution reconstruction platform for large-volume imaging and automated segmentation of neurons and synapses with molecular identity information. We used this platform to map inhibitory synaptic input fields of On-Off direction-selective ganglion cells (On-Off DSGCs), which are important for computing visual motion direction in the mouse retina. The reconstructions of On-Off DSGCs showed a GABAergic, receptor subtype-specific input field for generating direction selective responses without significant glycinergic inputs for mediating monosynaptic crossover inhibition. These results demonstrate unique capabilities of this super-resolution platform for interrogating neural circuitry.

Structural and molecular bases of rod photoreceptor morphogenesis and disease

“…6). A recent 4-color 3D STORM study of synaptic input fields in retinal interneurons confirmed the suitability of mouse retinal samples for such studies (Sigal et al, 2015). …”

Super‐resolution imaging of subcortical white matter using stochastic optical reconstruction microscopy (STORM) and super‐resolution optical fluctuation imaging (SOFI)

“…STORM imaging of Bassoon and the glutamate receptors GluR1 and GluR2 was also achieved in 4% paraformaldehyde‐fixed mouse brain tissue . A combination of immunohistochemistry for the synaptic scaffolding protein gephyrin, with transgenic expression of the fluorophores GFP and YFP, was performed in mouse retinal sections . This permitted detailed structural imaging of pre‐and postsynaptic domains in glycinergic and GABAergic synapses within the retina.…”

“…This permitted detailed structural imaging of pre‐and postsynaptic domains in glycinergic and GABAergic synapses within the retina. The tissue preparation protocol included refinements such as post‐fixing with glutaraldehyde and epoxy resin embedding followed by ultra‐thin sectioning (70 nm) . Similar protocols have been used for mitochondrial labelling in 10–20 μm sections of mouse brain, heart and kidney .…”

Mapping the connectivity of serotonin transporter immunoreactive axons to excitatory and inhibitory neurochemical synapses in the mouse limbic brain

“…Recently, improved automated or semi-automated techniques have been developed for in vitro and in vivo quantification of synapses using fluorescence microscopy (Ippolito and Eroglu 2010; Schätzle et al 2012; Dumitriu et al 2012; Busse and Smith 2013; Fogarty et al 2013; Danielson and Lee 2014; Sanders et al 2015; Klenowski et al 2015; Sigal et al 2015). The combination of fluorescence microscopy, immunolabeling of key pre- and postsynaptic markers of excitatory/inhibitory synapses and automated software analysis, has afforded new high-throughput methodology allowing for rapid quantification of putative neurochemical synapses.…”