A simple and reliable method for claustrum localization across age in mice.

The anatomical organization of the rodent claustrum remains obscure due to lack of clear borders that distinguish it from neighboring forebrain structures. Defining what constitutes the claustrum is imperative for elucidating its functions. Methods based on gene/protein expression or transgenic mice have been used to spatially outline the claustrum but often report incomplete labeling and/or lack of specificity during certain neurodevelopmental timepoints. To reliably identify claustrum projection cells in mice, we propose a simple immunolabelling method that juxtaposes the expression pattern of claustrum-enriched and cortical-enriched markers. We determined that claustrum cells immunoreactive for the claustrum-enriched markers Nurr1 and Nr2f2 are devoid of the cortical marker Tle4, which allowed us to differentiate the claustrum from adjoining cortical cells. Using retrograde tracing, we verified that nearly all claustrum projection neurons lack Tle4 but expressed Nurr1/Nr2f2 markers to different degrees. At neonatal stages between 7 and 21 days, claustrum projection neurons were identified by their Nurr1-postive/Tle4-negative expression profile, a time-period when other immunolabelling techniques used to localize the claustrum in adult mice are ineffective. Finally, exposure to environmental novelty enhanced the expression of the neuronal activation marker c-Fos in the claustrum region. Notably, c-Fos labeling was mainly restricted to Nurr1-positive cells and nearly absent from Tle4-positive cells, thus corroborating previous work reporting novelty-induced claustrum activation. Taken together, this method will aid in studying the claustrum during postnatal development and may improve histological and functional studies where other approaches are not amenable.

Brain-state-dependent constraints on claustrocortical communication and function.

Neural activity in the claustrum has been associated with a range of vigilance states, yet the activity patterns and efficacy of synaptic communication of identified claustrum neurons have not been thoroughly determined. Here, we show that claustrum neurons projecting to the retrosplenial cortex are most active during synchronized cortical states such as non-rapid eye movement (NREM) sleep and are suppressed during increased cortical desynchronization associated with arousal, movement, and REM sleep. The efficacy of claustrocortical signaling is increased during NREM and diminished during movement due in part to increased cholinergic tone. Finally, claustrum activation during NREM sleep enhances memory consolidation through the phase resetting of cortical delta waves. Therefore, claustrocortical communication is constrained to function most effectively during cognitive processes associated with synchronized cortical states, such as memory consolidation.

Topographic gradients define the projection patterns of the claustrum core and shell in mice.

The claustrum is densely connected to the cortex and participates in brain functions such as attention and sleep. Although some studies have reported the widely divergent organization of claustrum projections, others describe parallel claustrocortical connections to different cortical regions. Therefore, the details underlying how claustrum neurons broadcast information to cortical networks remain incompletely understood. Using multicolor retrograde tracing we determined the density, topography, and co-projection pattern of 14 claustrocortical pathways, in mice. We spatially registered these pathways to a common coordinate space and found that the claustrocortical system is topographically organized as a series of overlapping spatial modules, continuously distributed across the dorsoventral claustrum axis. The claustrum core projects predominantly to frontal-midline cortical regions, whereas the dorsal and ventral shell project to the cortical motor system and temporal lobe, respectively. Anatomically connected cortical regions receive common input from a subset of claustrum neurons shared by neighboring modules, whereas spatially separated regions of cortex are innervated by different claustrum modules. Therefore, each output module exhibits a unique position within the claustrum and overlaps substantially with other modules projecting to functionally related cortical regions. Claustrum inhibitory cells containing parvalbumin, somatostatin, and neuropeptide Y also show unique topographical distributions, suggesting different output modules are controlled by distinct inhibitory circuit motifs. The topographic organization of excitatory and inhibitory cell types may enable parallel claustrum outputs to independently coordinate distinct cortical networks.