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.

The claustrum (CLA) is a brain nucleus wedged between the cortex and striatum.The behaviors it has been implicated in include consciousness, attention, memory and salience detection; dysfunction of CLA circuits is associated with schizophrenia, epilepsy, parkinsonism and disrupted consciousness.While previous research has focused on the gross anatomy of the CLA, it is the functional communication of the CLA with other brain regions that generates behavioral output. Understanding CLA functional connectivity will bring us closer to understanding how the CLA is involved in different behaviors and how these dysfunctions can be remedied.The anterior cingulate cortex-projecting (CLA-ACC) neuron population was used as a model to investigate claustrocortical synaptic transmission. This thesis proposes that the CLA is organized as a highway for connections between brain regions. Paper I revealed that the CLA is organized as functional modules. Specifically, it showed that CLA-ACC neurons receive multicortical input biased towards frontal & limbic cortices rather than sensory cortices, and that CLA-ACC neurons could be segmented into at least two cortical targeting systems. An insular-claustrumanterior cingulate cortex circuit, which may be the substrate underpinning the Salience Network, was also identified. These findings support feedforward inhibition as a mechanism of action within the CLA.Paper II extended the concept of topological selectivity in the CLA to the singlecell level. Topological selectivity was previously known to exist at a population level. Characterization of the intrinsic electrophysiological properties of individual CLA-ACC neurons revealed four types of CLA-ACC populations. These CLA-ACC neurons were distributed heterogeneously with one type predominant in the anterior and posterior CLA and a second type prominent in the middle of the CLA.Paper III identified the cell-type and layer-specific cortical targets of the CLA. It showed that CLA-ACC neurons provide excitatory monosynaptic input to all layers of the ACC and that different neuron populations receive CLA input in a layer-dependent fashion. From these data, Paper III derived a scheme of CLA targets within a cortex.The findings from this thesis can be summarized using a transportation analogy.Although commonly described as a hub for cortical inputs and outputs, the CLA is likely organized as a collection of highways. A significantly large input should arrive within a small time-window to generate action potentials and enable downstream signal propagation. This is akin to a toll booth with a high toll fee that must be paid-in-full, without delays, before a vehicle can pass through. Projection neurons directed to the same cortical region may have different cell/layer targets. This is comparable to different vehicles on the same highway ending up in different destinations.The findings in this thesis add to our understanding of CLA functional organization by suggesting that any input received by the CLA must be sufficiently strong in order to overc...