![]() EM is a powerful tool that offers unparalleled nanometer resolution 3, sufficient to observe synaptic structures. The postsynaptic compartment contains a region that is protein dense called the postsynaptic density (PSD), which is comprised of neurotransmitter receptors, scaffolding proteins, and signaling molecules 2.ĭue to their small size, neuroscientists have relied on electron microscopy (EM) to observe chemical synapses and map connections between neurons. The presynaptic compartment contains a region that mediates neurotransmitter release called the active zone 1. They are located where presynaptic and postsynaptic neurons touch, and are composed of structures spanning hundreds of nanometers in size. Neurons are connected to each other primarily through chemical synapses. A comprehensive wiring diagram of neuronal connections is called a connectome, while the pursuit of a connectome is known as connectomics. Understanding these wiring patterns is a fundamental piece of the puzzle toward understanding how our brains work. Mammalian brains are extraordinarily complex pieces of circuitry, composed of trillions of connections between diverse cell types. We envision that spectral connectomics can be applied routinely in neurobiology labs to gain insights into normal and pathophysiological neuroanatomy. Furthermore, we show that correlative Brainbow and endogenous synaptic machinery immunostaining can define putative synaptic connections between neurons, as well as map putative inhibitory and excitatory inputs. We apply this strategy to directly link inhibitory neuron cell types with their morphologies. We combine multicolor labeling (Brainbow) of neurons with multi-round immunostaining Expansion Microscopy (miriEx) to simultaneously interrogate morphology, molecular markers, and connectivity in the same brain section. To address this, we devise a light microscopy approach for connectivity analysis of defined cell types called spectral connectomics. However, molecular information that specifies cell types is often lost in EM reconstructions. Electron microscopy (EM) has been the gold standard for connectivity analysis because nanoscale resolution is necessary to unambiguously resolve synapses. Mapping neuroanatomy is a foundational goal towards understanding brain function.
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