RECONSTRUCTION OF AXONAL COLLATERALS FROM SPARSE LOCUS COERULEUS NORADRENERGIC (LC-NA) NEURONS REVEALS THEIR MODULAR AXON PROJECTIONS

Noradrenergic (NA) neurons in the locus coeruleus (LC) have highly divergent axonal projections and play a critical role in behavior by supplying norepinephrine throughout the brain. Recent evidence suggests that the cortical projections of LC-NA neurons are organized into modules that coordinate various brain functions. However, the organization of these modules is not well understood, partly due to the limited axonal collateral data available from sparsely labeled neurons. Using TH-Cre mice, we reconstructed the axonal collaterals of a few dozen LC-NA neurons that project to the cingulate cortex (CgC). Retrograde AAV was injected into the CgC to drive Cre-dependent Flp expression. Then, AAV was injected into the LC for Flp-dependent GCaMP expression. Centered log-ratio transformation reveals a non-uniform axonal projection pattern, with several forebrain regions exhibiting marked enrichment of fiber density relative to the geometric mean of all others. Notably, these enriched regions—the rostrosplenial cortex, dorsal hippocampus, somatosensory cortex, and CgC (the rAAV injection site)—are functionally linked to navigation. To validate the analysis, we injected rAAV into the ventral hippocampus, which is not functionally linked to navigation, and observed a distinct NA fiber distribution pattern. Specifically, none of the enrichment detected from the CgC injection was found, except in the dorsal hippocampus. Instead, regions within the hypothalamus and thalamus exhibited relative enrichment of NA fibers. Together, these results demonstrate that LC-NA neurons are organized into distinct projection modules, each comprising a small number of neurons with a preference for specific forebrain targets that are functionally correlated.