Large-scale connectomics requires dense staining of neuronal tissue blocks for electron microscopy (EM). Here we report a large-volume dense en-bloc EM staining protocol that overcomes the staining gradients, which so far substantially limited the reconstructable volumes in three-dimensional (3D) EM. Our protocol provides densely reconstructable tissue blocks from mouse neocortex sized at least 1 mm in diameter. By relaxing the constraints on precise topographic sample targeting, it makes the correlated functional and structural analysis of neuronal circuits realistic.
Brain circuits in the neocortex develop from diverse types of neurons that migrate and form synapses. Here we quantify the circuit patterns of synaptogenesis for inhibitory interneurons in the developing mouse somatosensory cortex. We studied synaptic innervation of cell bodies, apical dendrites, and axon initial segments using three-dimensional electron microscopy focusing on the first 4 weeks postnatally (postnatal days P5 to P28). We found that innervation of apical dendrites occurs early and specifically: Target preference is already almost at adult levels at P5. Axons innervating cell bodies, on the other hand, gradually acquire specificity from P5 to P9, likely via synaptic overabundance followed by antispecific synapse removal. Chandelier axons show first target preference by P14 but develop full target specificity almost completely by P28, which is consistent with a combination of axon outgrowth and off-target synapse removal. This connectomic developmental profile reveals how inhibitory axons in the mouse cortex establish brain circuitry during development.
In mammals, sensory signals are transmitted via the thalamus primarily to layer 4 of the primary sensory cortices. While information about average neuronal connectivity in this layer is available, the detailed and higher-order circuit structure is not known. Here, we used 3-dimensional electron microscopy for a connectomic analysis of the thalamus-driven inhibitory network in a layer 4 barrel. We find that thalamic input drives a subset of interneurons with high specificity. These interneurons in turn target spiny stellate and star pyramidal excitatory neurons with subtype specificity. In addition, they create a directed disinhibitory network directly driven by the thalamic input. Together, this circuit can create differential windows of opportunity for activation of the types of excitatory neurons in dependence of strength and timing of thalamic input. With this, we have identified a so-far unknown degree of specialization of the microcircuitry in the main thalamocortical recipient layer of the primary sensory cortex.
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