Ultrastructure of synaptic connectivity within sub-regions of the suprachiasmatic nucleus revealed by a genetically encoded tag and Serial Blockface Electron Microscopy.

The hypothalamic suprachiasmatic nucleus (SCN) is the central circadian pacemaker in vertebrates. The SCN receives photic information exclusively through melanopsin-expressing retinal ganglion cells (mRGC) to synchronize circadian rhythms with the environmental light cycles. The SCN is composed of two major peptidergic neuron types in the core and shell regions of the SCN. Determining how mRGCs interact with the network of synaptic connections onto and between SCN neurons is key to understand how light regulates the circadian clock and to elucidate the relevant local circuits within the SCN. To map these connections, we used a newly developed Cre-dependent electron microscopy reporter, APEX2, to label the mitochondria of mRGC axons. Serial blockface scanning electron microscopy was then used to resolve the fine 3D structure of mRGC axons and synaptic boutons in the SCN of a male mouse. The resulting maps reveal patterns of connectomic organization in the core and shell of the SCN. We show that these regions are composed of different neuronal subtypes and differ with regard to the pattern of mRGC input, as the shell receives denser mRGC synaptic input compared to the core. This finding challenges the present view that photic information coming directly from the retina is received primarily by the core region of the SCN. Signification statement The hypothalamic suprachiasmatic nucleus (SCN) in the vertebrate brain serves as the central pacemaker regulating circadian rhythm throughout the body and as the principal hub for entraining circadian rhythm with the ambient light:dark cycle. Cellular and molecular studies have suggested heterogeneity of the SCN neurons and their connectivity, yet an ultrastructural characterization of the SCN connectivity is still lacking. In order to systematically investigate the connectivity within the SCN, we used a recently developed Cre-dependant electron microscopy reporter, APEX2, to specifically label mitochondria of mRGCs, and SBEM to produce image volumes of the two functional sub-regions of the SCN, the core and the shell. Our findings unveil several differences between the sub-regions, including synaptic input (retinal and non-retinal), the density of network of dendrites forming dendro-dendritic synapses. In addition, we established morphological criteria for discriminating between different types of axonic boutons.