The widespread reciprocal connectivity between the claustrum and the neocortex has stimulated numerous hypotheses regarding its function; all of these suggest that the claustrum acts as a hub that connects multiple cortical regions via dense reciprocal synaptic pathways. Although the connectivity between the anterior cingulate cortex (ACC) and the claustrum has been proposed as an important pathway for top-down cognitive control, little is known about the synaptic inputs that drive claustrum cells projecting to the ACC. Here, we used multi-neuron patch clamp recordings, retrograde and anterograde viral labeling, and optogenetics in mouse claustrum to investigate cortical inputs and outputs of ACC-projecting claustrum (CLA-ACC) neurons. Both ipsilateral and contralateral cortical regions were found to provide synaptic input to CLA-ACC neurons. These cortical regions were predominantly frontal and limbic regions and not primary sensorimotor regions. We show that CLA-ACC neurons receive monosynaptic input from the insular cortex, thereby revealing a potential claustrum substrate mediating the Salience Network. In contrast, sensorimotor cortical regions preferentially targeted non CLA-ACC claustrum neurons. Using dual retrograde labeling of claustrum projection neurons, we show selectivity also in the cortical targets of CLA-ACC neurons: whereas CLA-ACC neurons coprojected mainly to other frontal regions, claustrum neurons projecting to primary sensorimotor cortices selectively targeted other sensorimotor regions. Our results show that both cortical inputs to and projections from CLA-ACC neurons are highly selective, suggesting an organization of cortico-claustral connectivity into functional modules that could be specialized for processing different types of information.

Objective: To define the physiological properties of neurons projecting from the claustrum to the anterior cingulate cortex (ACC). Design: To identify the claustrum in live slices, we used a transgenic mouse line that expresses yellow fluorescent protein (YFP)-tagged Volvox channelrhodopsin-1 at high levels within the claustrum. Claustrum cells projecting to the ACC were identified by retrograde labelling. Whole-cell patch-clamp recordings from labelled claustrum neurons were used to characterize the intrinsic electrical properties of these neurons. Cells were classified according to their intrinsic electrical properties, based on a previous classification scheme. Results: Labelled neurons were found in the claustrum but not the insular cortex. Four types of ACC-projecting neurons were identified based on action potential adaptation and waveform: strongly adapting (SA) cell types 2, 3 and 4, and moderately adapting (MA) cell type 2. Labelled cells were predominantly SA4 in the anterior (44%) and posterior (63%) claustrum, while MA2 predominated (77%) in the central claustrum. The male anterior claustrum showed a bias toward SA3 cells (53%) while the female anterior claustrum showed a bias toward SA3 cells (76%). Conclusions: There is ipsilateral dominance for ACC-projecting claustrum neurons, with the intrinsic properties of these neurons varying along the anterior-posterior axis. Sexual dimorphism was observed in ACC-projecting claustrum cells. Our results are consistent with the hypothesis that the claustrum serves as a link between the insular cortex and the ACC. ARTICLE HISTORY

The claustrum (CLA) is a brain nucleus located between the insula and lateral striatum, implicated in a wide range of behaviors. Underpinning the different behavioral phenotypes is the connectivity between the claustrum and various cortical regions, including the anterior cingulate cortex (ACC). CLA projection neurons are glutamatergic neurons, however, the impact of CLA on its cortical targets has been shown in some studies to be inhibitory. Such inhibition is likely to arise from claustral activation of cortical interneurons, however, the intricate synaptic connectivity between different CLA and cortical cell types is not known. Here, we combine in vivo and ex vivo electrophysiology and optogenetics to reveal the functional organization of the CLA-ACC pathway according to the identity of its pre- and postsynaptic populations. Optogenetic stimulation of CLA neurons in awake mice resulted in multiphasic excitatory and inhibitory responses in ACC cells, which depended on the layer, cell type, and stimulated CLA population. Using ex vivo paired recordings in ACC, monosynaptic responses were recorded in pyramidal cells and different types of interneurons following photostimulation of CLA-ACC synaptic terminals. CLA axons formed monosynaptic connections in all ACC cortical layers, but the probability and strength of synaptic responses depended on the type of CLA projection, target layer in ACC, and the type of postsynaptic neuron. This intricate organization of the CLA-ACC pathway may explain the complex impact of CLA on ACC and other cortical regions, thus resolving some of the discrepancies in the field and shedding light on the functional role CLA plays in cortical function.