The claustrum is known for its extensive connectivity with many other forebrain regions, but its elongated shape and deep location have made further study difficult. We have sought to understand when mouse claustrum neurons are born, where they are located in developing brains, and when they develop their widespread connections to the cortex. We established that a well-characterized parvalbumin plexus, which identifies the claustrum in adults, is only present from postnatal day (P) 21. A myeloarchitectonic outline of the claustrum can be derived from a triangular fiber arrangement from P15. A dense patch of Nurr1+ cells is present at its core and is already evident at birth. Bromodeoxyuridine birth dating of forebrain progenitors reveals that the majority of claustrum neurons are born during a narrow time window centered on embryonic day 12.5, which is later than the adjacent subplate and endopiriform nucleus. Retrograde tracing revealed that claustrum projections to anterior cingulate (ACA) and retrosplenial cortex (RSP) follow distinct developmental trajectories. Claustrum–ACA connectivity matures rapidly and reaches adult-like innervation density by P10, whereas claustrum–RSP innervation emerges later over a protracted time window. This work establishes the timeline of claustrum development and provides a framework for understanding how the claustrum is built and develops its unique connectivity.

The claustrum is known for its extensive connectivity with many other forebrain regions, but its elongated shape and deep location have made further study difficult. We have sought to understand when mouse claustrum neurons are born, where they are located in developing brains and when they develop their widespread connections to cortex. We established that a well-characterised parvalbumin-plexus, which identifies the claustrum in adults, is only present from postnatal day (P)21. A myeloarchitectonic outline of the claustrum can be derived from a triangular fibre arrangement from P15. A dense patch of Nurr1+ cells is present at its core, and is already evident at birth. BrdU-birthdating of forebrain progenitors reveals that the majority of claustrum neurons are born during a narrow time window centred on embryonic day (E)12.5, which is later than the adjacent subplate and endopiriform nucleus. Retrograde tracing revealed that claustrum projections to anterior cingulate (ACA) and retrosplenial cortex (RSP) follow distinct developmental trajectories. Claustrum-ACA connectivity matures rapidly, and reaches adult-like innervation density by P10, whereas claustrum-RSP innervation emerges later over a protracted time window. This work establishes the timeline of claustrum development, and provides a framework for understanding how the claustrum is built and develops its unique connectivity.