Is REM sleep a paradoxical state?: Different neurons are activated in the cingulate cortices and the claustrum during wakefulness and paradoxical sleep hypersomnia.

Michel Jouvet proposed in 1959 that REM sleep is a paradoxical state since it was characterized by the association of a cortical activation similar to wakefulness (W) with muscle atonia. Recently, we showed using cFos as a marker of activity that cortical activation during paradoxical sleep (PS) was limited to a few limbic cortical structures in contrast to W during which all cortices were strongly activated. However, we were not able to demonstrate whether the same neurons are activated during PS and W and to rule out that the activation observed was not linked with stress induced by the flowerpot method of PS deprivation. In the present study, we answered to these two questions by combining tdTomato and cFos immunostaining in the innovative TRAP2 transgenic mice exposed one week apart to two periods of W (W-W mice), PS rebound (PSR-PSR) or a period of W followed by a period of PSR (W-PSR mice). Using such method, we showed that different neurons are activated during W and PSR in the anterior cingulate (ACA) and rostral and caudal retrosplenial (rRSP and cRSP) cortices as well as the claustrum (CLA) previously shown to contain a large number of activated neurons after PSR. Further, the distribution of the neurons during PSR in the rRSP and cRSP was limited to the superficial layers while it was widespread across all layers during W. Our results clearly show at the cellular level that PS and W are two completely different states in term of neocortical activation.

Synaptic Connectivity between the Cortex and Claustrum Is Organized into Functional Modules.

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 co-projected 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.

In search of common developmental and evolutionary origin of the claustrum and subplate.

The human claustrum, a major hub of widespread neocortical connections, is a thin, bilateral sheet of gray matter located between the insular cortex and the striatum. The subplate is a largely transient cortical structure that contains some of the earliest generated neurons of the cerebral cortex and has important developmental functions to establish intra- and extracortical connections. In human and macaque some subplate cells undergo regulated cell death, but some remain as interstitial white matter cells. In mouse and rat brains a compact layer is formed, Layer 6b, and it remains underneath the cortex, adjacent to the white matter. Whether Layer 6b in rodents is homologous to primate subplate or interstitial white matter cells is still debated. Gene expression patterns, such as those of Nurr1/Nr4a2, have suggested that the rodent subplate and the persistent subplate cells in Layer 6b and the claustrum might have similar origins. Moreover, the birthdates of the claustrum and Layer 6b are similarly precocious in mice. These observations prompted our speculations on the common developmental and evolutionary origin of the claustrum and the subplate. Here we systematically compare the currently available data on cytoarchitecture, evolutionary origin, gene expression, cell types, birthdates, neurogenesis, lineage and migration, circuit connectivity, and cell death of the neurons that contribute to the claustrum and subplate. Based on their similarities and differences we propose a partially common early evolutionary origin of the cells that become claustrum and subplate, a likely scenario that is shared in these cell populations across all amniotes.

A claustrum in reptiles and its role in slow-wave sleep.

The mammalian claustrum, owing to its widespread connectivity with other forebrain structures, has been hypothesized to mediate functions that range from decision-making to consciousness1. Here we report that a homologue of the claustrum, identified by single-cell transcriptomics and viral tracing of connectivity, also exists in a reptile-the Australian bearded dragon Pogona vitticeps. In Pogona, the claustrum underlies the generation of sharp waves during slow-wave sleep. The sharp waves, together with superimposed high-frequency ripples2, propagate to the entire neighbouring pallial dorsal ventricular ridge (DVR). Unilateral or bilateral lesions of the claustrum suppress the production of sharp-wave ripples during slow-wave sleep in a unilateral or bilateral manner, respectively, but do not affect the regular and rapidly alternating sleep rhythm that is characteristic of sleep in this species3. The claustrum is thus not involved in the generation of the sleep rhythm itself. Tract tracing revealed that the reptilian claustrum projects widely to a variety of forebrain areas, including the cortex, and that it receives converging inputs from, among others, areas of the mid- and hindbrain that are known to be involved in wake-sleep control in mammals4-6. Periodically modulating the concentration of serotonin in the claustrum, for example, caused a matching modulation of sharp-wave production there and in the neighbouring DVR. Using transcriptomic approaches, we also identified a claustrum in the turtle Trachemys scripta, a distant reptilian relative of lizards. The claustrum is therefore an ancient structure that was probably already present in the brain of the common vertebrate ancestor of reptiles and mammals. It may have an important role in the control of brain states owing to the ascending input it receives from the mid- and hindbrain, its widespread projections to the forebrain and its role in sharp-wave generation during slow-wave sleep.

Cytoarchitecture of the dorsal claustrum of the cat: a quantitative Golgi study.

The claustrum is a subcortical nucleus, found in the telencephalon of all placental mammals. Earlier Golgi studies have mostly focused on a qualitative description of the types of neurons. The aim of the present study was to describe the types of neurons found in the dorsal claustrum of the cat using the Golgi impregnation method and to perform a quantitative analysis of the following morphometric parameters: number of terminals (ends), total dendritic length, dendritic complexity, spine density (in spiny projection neurons), varicosity density (in aspiny interneurons). We used specimens from 5 healthy male cats stained according to the Golgi-Cox method. The dendritic trees of the studied neurons were then reconstructed through the Neurolucida software. Values of the studied quantitative parameters were obtained automatically and tested for statistically significant differences. Five types of spiny neurons were observed-large, medium-sized and small multipolar, bipolar and pyramidal-like. In addition, we described three types of aspiny neurons. The quantitative values and the statistical analysis were presented with tables and diagrams. In conclusion, we have presented a detailed analysis of the cytoarchitecture of the DC of the cat and have reported the first quantitative data on a number of morphometric parameters.

Input-output organization of the mouse claustrum.

Progress in determining the precise organization and function of the claustrum (CLA) has been hindered by the difficulty in reliably targeting these neurons. To overcome this, we used a projection-based targeting strategy to selectively label CLA principal neurons. Combined with adeno-associated virus (AAV) and monosynaptic rabies tracing techniques, we systematically examined the pre-synaptic input and axonal output of this structure. We found that CLA neurons projecting to retrosplenial cortex (RSP) collateralize extensively to innervate a variety of higher-order cortical regions. No subcortical labeling was found, with the exception of sparse terminals in the basolateral amygdala (BLA). This pattern of output was similar to cingulate- and visual cortex-projecting CLA neurons, suggesting a common targeting scheme among these projection-defined populations. Rabies virus tracing directly demonstrated widespread synaptic inputs to RSP-projecting CLA neurons from both cortical and subcortical areas. The strongest inputs arose from classically defined limbic regions, including medial prefrontal cortex, anterior cingulate, BLA, ventral hippocampus, and neuromodulatory systems such as the dorsal raphe and cholinergic basal forebrain. These results suggest that the CLA may integrate information related to the emotional salience of stimuli and may globally modulate cortical state by broadcasting its output uniformly across a variety of higher cognitive centers.