Capturing and Manipulating Activated Neuronal Ensembles with CANE Delineates a Hypothalamic Social-Fear Circuit

Abstract

SUMMARY We developed a technology (Capturing Activated Neural Ensembles, or CANE) to label, manipulate, and trans-synaptically trace neural circuits that are transiently activated in behavioral contexts with high efficiency and temporal precision. CANE consists of a knock-in mouse and engineered viruses designed to specifically infect activated neurons. Using CANE, we selectively labeled neurons that were activated by either fearful or aggressive social encounters in a hypothalamic subnucleus previously known as a locus for aggression, and discovered that social fear and aggression neurons are intermixed but largely distinct. Optogenetic stimulation of CANE-captured social fear neurons (SFNs) is sufficient to evoke fear-like behaviors in normal social contexts, whereas silencing SFNs resulted in reduced social avoidance. CANE-based mapping of axonal projections and presynaptic inputs to SFNs further revealed a highly distributed and recurrent neural network. CANE is a broadly applicable technology for dissecting causality and connectivity of spatially intermingled but functionally distinct ensembles.

Genetic strategies to access activated neurons

“…CANE-captured social fear-activated neurons (SFNs) in the ventromedial hypothalamus (VMHvl) bi-directionally controlled social fear. Because SFNs comprise only 3% of VMHvl neurons and specific molecular markers are not available, it would have been difficult to target them without activity-based methods (55). …”

Connectional architecture of a mouse hypothalamic circuit node controlling social behavior

“…However, these techniques are unable to distinguish genetically-defined cell populations within VMHvl, which is heterogeneous (9). Further, with a few exceptions (45,46) most studies report on a single type of tracing method (e.g., anterograde or retrograde), making it difficult to directly compare results across publications. To compare more directly the inputs and outputs of VMHvl Esr1 neurons, we used Esr1-Cre knock-in mice (7), together with Cre-dependent anterograde (12) and modified monosynaptic retrograde rabies (10,11) viruses (see Methods), stereotaxically injected into VMHvl, and analyzed the results using serial 2-photon tomography (Tissue-Cyte, (47) at 100 µm z-intervals with 0.35 µm x-y resolution.…”

“…First, we do not yet know the degree of neuronal subtype diversity among VMHvl Esr1 neurons, and its relationship to overall VMHvl connectional architecture. It is already clear from recently published work that the VMHvl contains subpopulations of neurons with social behavior22 functions distinct from those involved in aggression(9,46,59,60). Further, preliminary singlecell RNAseq analysis suggests that there are at least 4 transcriptomically distinct subsets of VMHvl Esr1 neurons, as well as ~12 other Esr1 -VMHvl cell populations (DWK and DJA, unpublished).…”

Identification of midbrain neurons essential for vocal communication

“…Therefore, a critical step to dissect the brain-wide 34 networks important for vocalization requires a method to selectively identify and manipulate 35 vocalization-related PAG neurons. To this end, we used a recently developed immediate early 36 gene Fos-based method (CANE 10,11 ; Fig. 1) to genetically label, identify, and manipulate PAG 37 neurons that are selectively active when male mice produce courtship ultrasonic vocalizations 38 (USVs), which comprise individual vocal elements (i.e., syllables) that are organized into 39 multisyllabic bouts that last from hundreds of milliseconds to tens of seconds in duration 14-19 40 ( Fig.…”