Sleep affects brain activity globally, but many cortical sleep waves are spatially confined. Local rhythms serve cortical area-specific sleep needs and functions; however, mechanisms controlling locality are unclear. We identify the thalamic reticular nucleus (TRN) as a source for local, sensory-cortex-specific non-rapid-eye-movement sleep (NREMS) in mouse. Neurons in optogenetically identified sensory TRN sectors showed stronger repetitive burst discharge compared to non-sensory TRN cells due to higher activity of the low-threshold Ca2+ channel CaV3.3. Major NREMS rhythms in sensory but not non-sensory cortical areas were regulated in a CaV3.3-dependent manner. In particular, NREMS in somatosensory cortex was enriched in fast spindles, but switched to delta wave-dominated sleep when CaV3.3 channels were genetically eliminated or somatosensory TRN cells chemogenetically hyperpolarized. Our data indicate a previously unrecognized heterogeneity in a powerful forebrain oscillator that contributes to sensory-cortex-specific and dually regulated NREMS, enabling local sleep regulation according to use- and experience-dependence.

Highlights d In NREMS, thalamic noradrenaline (NA) levels are higher than in quiet wakefulness d Thalamic NA fluctuates over 50 s and is anticorrelated to sleep spindles d NA released from LC depolarizes thalamic neurons through a1and b-adrenoceptors d Infraslow LC activity coordinates heart-rate variations with spindles

In spite of the uniform appearance of sleep as a behavior, the sleeping brain does not produce electrical activities in unison. Different types of brain rhythms arise during sleep and vary between layers, areas, or from one functional system to another. Local heterogeneity of such activities, here referred to as local sleep, overturns fundamental tenets of sleep as a globally regulated state. However, little is still known about the neuronal circuits involved and how they can generate their own specifically-tuned sleep patterns. NREM sleep patterns emerge in the brain from interplay of activity between thalamic and cortical networks. Within this fundamental circuitry, it now turns out that the thalamic reticular nucleus (TRN) acts as a key player in local sleep control. This is based on a marked heterogeneity of the TRN in terms of its cellular and synaptic architecture, which leads to a regional diversity of NREM sleep hallmarks, such as sleep spindles, delta waves and slow oscillations. This provides first evidence for a subcortical circuit as a determinant of cortical local sleep features. Here, we review novel cellular and functional insights supporting TRN heterogeneity and how these elements come together to account for local NREM sleep. We also discuss open questions arising from these studies, focusing on mechanisms of sleep regulation and the role of local sleep in brain plasticity and cognitive functions.

Highlights d dPreS/RS cortices send a monosynaptic glutamatergic projection to anterodorsal TRN cells d This projection leads to feedforward inhibition of anterior thalamic cells d Chemogenetic TRN silencing deteriorates thalamic headdirection cell tuning d Chemogenetic TRN silencing alters search strategies in the Morris water maze

Cell-penetrating peptides (CPPs) allow intracellular delivery of bioactive cargo molecules. The mechanisms allowing CPPs to enter cells are ill-defined. Using a CRISPR/Cas9-based screening, we discovered that KCNQ5, KCNN4, and KCNK5 potassium channels positively modulate cationic CPP direct translocation into cells by decreasing the transmembrane potential (Vm). These findings provide the first unbiased genetic validation of the role of Vm in CPP translocation in cells. In silico modeling and live cell experiments indicate that CPPs, by bringing positive charges on the outer surface of the plasma membrane, decrease the Vm to very low values (–150 mV or less), a situation we have coined megapolarization that then triggers formation of water pores used by CPPs to enter cells. Megapolarization lowers the free energy barrier associated with CPP membrane translocation. Using dyes of varying dimensions in CPP co-entry experiments, the diameter of the water pores in living cells was estimated to be 2 (–5) nm, in accordance with the structural characteristics of the pores predicted by in silico modeling. Pharmacological manipulation to lower transmembrane potential boosted CPP cellular internalization in zebrafish and mouse models. Besides identifying the first proteins that regulate CPP translocation, this work characterized key mechanistic steps used by CPPs to cross cellular membranes. This opens the ground for strategies aimed at improving the ability of cells to capture CPP-linked cargos in vitro and in vivo.

Thalamus and cortex represent a highly integrated processing unit that elaborates sensory representations. Interposed between cortex and thalamus, the nucleus Reticularis thalami (nRt) receives strong cortical glutamatergic input and mediates top-down inhibitory feedback to thalamus. Despite growing appreciation that the nRt is integral for thalamocortical functions from sleep to attentional wakefulness, we still face considerable gaps in the synaptic bases for cortico-nRt communication and plastic regulation. Here, we examined modulation of nRt excitability by cortical synaptic drive in Ntsr1-Cre x ChR2tg/+ mice expressing Channelrhodopsin2 in layer 6 corticothalamic cells. We found that cortico-nRt synapses express a major portion of NMDA receptors containing the GluN2C subunit (GluN2C-NMDARs). Upon repetitive photoactivation (10 Hz trains), GluN2C-NMDARs induced a long-term increase in nRt excitability involving a potentiated recruitment of T-type Ca2+ channels. In anaesthetized mice, analogous stimulation of cortical afferents onto nRt produced long-lasting changes in cortical local field potentials (LFPs), with delta oscillations being augmented at the expense of slow oscillations. This shift in LFP spectral composition was sensitive to NMDAR blockade in the nRt. Our data reveal a novel mechanism involving plastic modification of synaptically recruited T-type Ca2+ channels and nRt bursting and indicate a critical role for GluN2C-NMDARs in thalamocortical rhythmogenesis.

30Cell-penetrating peptides (CPPs) allow intracellular delivery of cargo molecules. CPPs 31 provide efficient methodology to transfer bioactive molecules in cells, in particular in 32 conditions when transcription or translation of cargo-encoding sequences is not 33 desirable or achievable. The mechanisms allowing CPPs to enter cells are ill-defined 34 and controversial. This work identifies potassium channels as key regulators of cationic 35 CPP translocation. Using a CRISPR/Cas9-based screening, we discovered that 36 KCNQ5, KCNN4, and KCNK5 positively modulate CPP cellular direct translocation by 37 reducing transmembrane potential (Vm). Cationic CPPs further decrease the Vm to 38 megapolarization values (about -150 mV) leading to the formation of ~2 nm-wide water 39 pores used by CPPs to access the cell's cytoplasm. Pharmacological manipulation to 40 lower transmembrane potential boosted CPPs cellular uptake in zebrafish and mouse 41 models. Besides identifying the first genes that regulate CPP translocation, this work 42 characterizes key mechanistic steps used by CPPs to cross cellular membrane. This 43 opens the ground for pharmacological strategies augmenting the susceptibility of cells 44 to capture CPP-linked cargos in vitro and in vivo. 45 46Cell-penetrating peptides (CPPs) are non-toxic molecules of 5-30 amino acids that can 47 translocate into living cells. CPPs can be hooked to a variety of cargos (siRNAs, DNA, 48 polypeptides, liposomes, nanoparticles, etc.) to allow their transport into cells for 49 therapeutic or experimental purposes (1-10). The origin of CPPs is diverse. For 50 example, TAT48-57 is a 10 amino-acid fragment derived from the trans-activator of 51 transcription (TAT) HIV-1 protein (11, 12), penetratin is a 16 amino-acid peptide 52 derived from the Antennapedia Drosophila melanogaster protein (13), and MAP (model 53 amphipatic peptide) is a synthetic alanine/leucine/lysine-rich peptide (14). The vast 54 majority of CPPs are cationic (1, 3, 6, 7). 55How CPPs enter cells is debated and not fully characterized at the molecular level 56 (reviewed in (1-8)). Due to this knowledge gap, it is difficult to ameliorate CPP cellular 57 entry and this slows down development of CPP-based interventions. Two main modes 58 of CPP entry have been described: endocytosis and direct translocation(1-8). 59Endocytosed CPPs gain cytosolic access by escaping endosomes. Direct 60 translocation has been proposed to occur through transient pore formation or 61 membrane destabilization. Endocytosis and direct translocation are not mutually 62 exclusive. Several entry routes can be followed simultaneously by a given CPP in a 63 given cell line (9, 10). 64Here, we used CRISPR/Cas9-screenings to identify proteins required for the cellular 65 uptake of CPPs. This approach identified three potassium channels as mandatory for 66 the direct translocation of CPPs into various cell types. Further, we highlighted the 67 requirement of an appropriate membrane potential to generate a 2 nm-wide water 68 pores through which...

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