Determination of the molecular architecture of synapses requires nanoscopic image resolution and specific molecular recognition, a task that has so far defied many conventional imaging approaches. Here we present a super-resolution fluorescence imaging method to visualize the molecular architecture of synapses in the brain. Using multicolor, three-dimensional stochastic optical reconstruction microscopy, the distributions of synaptic proteins can be measured with nanometer precision. Furthermore, the wide-field, volumetric imaging method enables high-throughput, quantitative analysis of a large number of synapses from different brain regions. To demonstrate the capabilities of this approach, we have determined the organization of ten protein components of the presynaptic active zone and the postsynaptic density. Variations in synapse morphology, neurotransmitter receptor composition, and receptor distribution were observed both among synapses and across different brain regions. Combination with optogenetics further allowed molecular events associated with synaptic plasticity to be resolved at the single-synapse level.

Mice display robust, stereotyped behaviors toward pups: virgin males typically attack pups, while virgin females and sexually experienced males and females display parental care. We show here that virgin males genetically impaired in vomeronasal sensing do not attack pups and are parental. Further, we uncover a subset of galanin-expressing neurons in the medial preoptic area (MPOA) that are specifically activated during male and female parenting, and a different subpopulation activated during mating. Genetic ablation of MPOA galanin neurons results in dramatic impairment of parental responses in males and females and affects male mating. Optogenetic activation of these neurons in virgin males suppresses inter-male and pup-directed aggression and induces pup grooming. Thus, MPOA galanin neurons emerge as an essential regulatory node of male and female parenting behavior and other social responses. These results provide an entry point to a circuit-level dissection of parental behavior and its modulation by social experience.

Combining rabies-virus tracing, optical clearing (CLARITY), and whole-brain light-sheet imaging, we mapped the monosynaptic inputs to midbrain dopamine neurons projecting to different targets (different parts of the striatum, cortex, amygdala, etc) in mice. We found that most populations of dopamine neurons receive a similar set of inputs rather than forming strong reciprocal connections with their target areas. A common feature among most populations of dopamine neurons was the existence of dense ‘clusters’ of inputs within the ventral striatum. However, we found that dopamine neurons projecting to the posterior striatum were outliers, receiving relatively few inputs from the ventral striatum and instead receiving more inputs from the globus pallidus, subthalamic nucleus, and zona incerta. These results lay a foundation for understanding the input/output structure of the midbrain dopamine circuit and demonstrate that dopamine neurons projecting to the posterior striatum constitute a unique class of dopamine neurons regulated by different inputs.DOI: http://dx.doi.org/10.7554/eLife.10032.001