Kinase signalling in excitatory neurons regulates sleep quantity and depth.

Progress has been made in the elucidation of sleep and wakefulness regulation at the neurocircuit level1,2. However, the intracellular signalling pathways that regulate sleep and the neuron groups in which these intracellular mechanisms work remain largely unknown. Here, using a forward genetics approach in mice, we identify histone deacetylase 4 (HDAC4) as a sleep-regulating molecule. Haploinsufficiency of Hdac4, a substrate of salt-inducible kinase 3 (SIK3)3, increased sleep. By contrast, mice that lacked SIK3 or its upstream kinase LKB1 in neurons or with a Hdac4S245A mutation that confers resistance to phosphorylation by SIK3 showed decreased sleep. These findings indicate that LKB1-SIK3-HDAC4 constitute a signalling cascade that regulates sleep and wakefulness. We also performed targeted manipulation of SIK3 and HDAC4 in specific neurons and brain regions. This showed that SIK3 signalling in excitatory neurons located in the cerebral cortex and the hypothalamus positively regulates EEG delta power during non-rapid eye movement sleep (NREMS) and NREMS amount, respectively. A subset of transcripts biased towards synaptic functions was commonly regulated in cortical glutamatergic neurons through the expression of a gain-of-function allele of Sik3 and through sleep deprivation. These findings suggest that NREMS quantity and depth are regulated by distinct groups of excitatory neurons through common intracellular signals. This study provides a basis for linking intracellular events and circuit-level mechanisms that control NREMS.

Remapping of Adult-Born Neuron Activity during Fear Memory Consolidation in Mice.

The mammalian hippocampal dentate gyrus is a unique memory circuit in which a subset of neurons is continuously generated throughout the lifespan. Previous studies have shown that the dentate gyrus neuronal population can hold fear memory traces (i.e., engrams) and that adult-born neurons (ABNs) support this process. However, it is unclear whether ABNs themselves hold fear memory traces. Therefore, we analyzed ABN activity at a population level across a fear conditioning paradigm. We found that fear learning did not recruit a distinct ABN population. In sharp contrast, a completely different ABN population was recruited during fear memory retrieval. We further provide evidence that ABN population activity remaps over time during the consolidation period. These results suggest that ABNs support the establishment of a fear memory trace in a different manner to directly holding the memory. Moreover, this activity remapping process in ABNs may support the segregation of memories formed at different times. These results provide new insight into the role of adult neurogenesis in the mammalian memory system.

Synthesis and xanthine oxidase inhibitory activity of 5,6-dihydropyrazolo/pyrazolo[1,5-c]quinazoline derivatives.

Some 5,6-dihydropyrazolo/pyrazolo[1,5-c]quinazoline derivatives were rationally designed, synthesized and evaluated for in vitro xanthine oxidase inhibitory activity for the first time. Some notions about structure activity relationships are presented. The compounds 6g, 6h and 6e were found to be significantly active against XO. The compound 6g emerged as the most potent XO inhibitor as compared to allopurinol and free radical scavenger. The molecular docking of 6g into the XO active site highlighted its mode of binding and important interactions such as hydrogen bonding, π-π stacking with amino acid residues like Ser876, Thr1010, Phen914, Phe1009 and Phe649 and its close proximity to dioxothiomolybdenum (MOS).

Warm sensitive neurons of the preoptic area regulate ambient temperature related changes in sleep in the rat.

Warm sensitive neurons (WSN) play a major role not only in body temperature regulation, but also in sleep regulation. The present study was undertaken to investigate the role of WSN of the preoptic area (POA) in mediating the ambient temperature (T(amb)) related changes in sleep. The effect of T(amb) changes on sleep and body temperature was studied in rats before and after destruction of WSN of the POA by local intracerebral injection of capsaicin. Though the rats preferred 27 degrees C T(amb), they slept maximum at 30 degrees C. After destruction of WSN of the POA, slow wave sleep (SWS) peak was brought down to 27 degrees C, which was the preferred T(amb) of the rats. This indicates that WSN of the POA mediate the increase in SWS, at temperatures higher than preferred T(amb). On the other hand, in WSN destroyed rats, rapid eye movement (REM) sleep was maximum at 33 degrees C. It suggests that the REM sleep generation is under inhibitory control of the WSN of the POA. The study supports several earlier reports that the neurons of the POA play a key role in coordinating sleep and body temperature regulation.

Real-time, automatic, open-source sleep stage classification system using single EEG for mice.

We developed a real-time sleep stage classification system with a convolutional neural network using only a one-channel electro-encephalogram source from mice and universally available features in any time-series data: raw signal, spectrum, and zeitgeber time. To accommodate historical information from each subject, we included a long short-term memory recurrent neural network in combination with the universal features. The resulting system (UTSN-L) achieved 90% overall accuracy and 81% multi-class Matthews Correlation Coefficient, with particularly high-quality judgements for rapid eye movement sleep (91% sensitivity and 98% specificity). This system can enable automatic real-time interventions during rapid eye movement sleep, which has been difficult due to its relatively low abundance and short duration. Further, it eliminates the need for ordinal pre-calibration, electromyogram recording, and manual classification and thus is scalable. The code is open-source with a graphical user interface and closed feedback loop capability, making it easily adaptable to a wide variety of end-user needs. By allowing large-scale, automatic, and real-time sleep stage-specific interventions, this system can aid further investigations of the functions of sleep and the development of new therapeutic strategies for sleep-related disorders.

Calcium imaging of adult-born neurons in freely moving mice.

Adult-born neurons (ABNs) in the dentate gyrus bestow unique cellular plasticity to the mammalian brain. We recently found that the activity of ABNs during sleep is necessary for memory consolidation. Here, we describe our method for Ca2+ imaging of ABN activity using a miniaturized fluorescent microscope and sleep recordings. As preparatory surgery and post-recording data processing can be major obstacles, we provide detailed descriptions and problem-solving tips. For complete details on the use and execution of this protocol, please refer to Kumar et al. (2020).

Sparse Activity of Hippocampal Adult-Born Neurons during REM Sleep Is Necessary for Memory Consolidation.

The occurrence of dreaming during rapid eye movement (REM) sleep prompts interest in the role of REM sleep in hippocampal-dependent episodic memory. Within the mammalian hippocampus, the dentate gyrus (DG) has the unique characteristic of exhibiting neurogenesis persisting into adulthood. Despite their small numbers and sparse activity, adult-born neurons (ABNs) in the DG play critical roles in memory; however, their memory function during sleep is unknown. Here, we investigate whether young ABN activity contributes to memory consolidation during sleep using Ca2+ imaging in freely moving mice. We found that contextual fear learning recruits a population of young ABNs that are reactivated during subsequent REM sleep against a backdrop of overall reduced ABN activity. Optogenetic silencing of this sparse ABN activity during REM sleep alters the structural remodeling of spines on ABN dendrites and impairs memory consolidation. These findings provide a causal link between ABN activity during REM sleep and memory consolidation.

Effect of context exposure after fear learning on memory generalization in mice.

BACKGROUND: The conditions under which memory generalization occurs are not well understood. Although it is believed that fear memory generalization is gradually established after learning, it is not clear whether experiences soon after learning affect generalization. RESULTS: Using a contextual fear conditioning paradigm in mice, we found that fear memory generalization occurred when mice were exposed to a familiar, unconditioned context soon after fear learning. CONCLUSIONS: Our results suggest that the familiarity of contexts and the timing of their exposure influences memory generalization, which increases our understanding of the mechanisms of generalization.

Cacna1c (Cav1.2) Modulates Electroencephalographic Rhythm and Rapid Eye Movement Sleep Recovery.

STUDY OBJECTIVES: The CACNA1C gene encodes the alpha 1C (α1C) subunit of the Cav1.2 voltage-dependent L-type calcium channel (LTCC). Some of the other voltage-dependent calcium channels, e.g., P-/Q-type, Cav2.1; N-type, Cav2.2; E-/R-type, Cav2.3; and T-type, Cav3.3 have been implicated in sleep modulation. However, the contribution of LTCCs to sleep remains largely unknown. Based on recent genome-wide association studies, CACNA1C emerged as one of potential candidate genes associated with both sleep and psychiatric disorders. Indeed, most patients with mental illnesses have sleep problems and vice versa. DESIGN: To investigate an impact of Cav1.2 on sleep-wake behavior and electroencephalogram (EEG) activity, polysomnography was performed in heterozygous Cacna1c (HET) knockout mice and their wild-type (WT) littermates under baseline and challenging conditions (acute sleep deprivation and restraint stress). MEASUREMENTS AND RESULTS: HET mice displayed significantly lower EEG spectral power than WT mice across high frequency ranges (beta to gamma) during wake and rapid eye movement (REM) sleep. Although HET mice spent slightly more time asleep in the dark period, daily amounts of sleep did not differ between the two genotypes. However, recovery sleep after exposure to both types of challenging stress conditions differed markedly; HET mice exhibited reduced REM sleep recovery responses compared to WT mice. CONCLUSIONS: These results suggest the involvement of Cacna1c (Cav1.2) in fast electroencephalogram oscillations and REM sleep regulatory processes. Lower spectral gamma activity, slightly increased sleep demands, and altered REM sleep responses found in heterozygous Cacna1c knockout mice may rather resemble a sleep phenotype observed in schizophrenia patients.

Inhibiting the Activity of CA1 Hippocampal Neurons Prevents the Recall of Contextual Fear Memory in Inducible ArchT Transgenic Mice.

The optogenetic manipulation of light-activated ion-channels/pumps (i.e., opsins) can reversibly activate or suppress neuronal activity with precise temporal control. Therefore, optogenetic techniques hold great potential to establish causal relationships between specific neuronal circuits and their function in freely moving animals. Due to the critical role of the hippocampal CA1 region in memory function, we explored the possibility of targeting an inhibitory opsin, ArchT, to CA1 pyramidal neurons in mice. We established a transgenic mouse line in which tetracycline trans-activator induces ArchT expression. By crossing this line with a CaMKIIα-tTA transgenic line, the delivery of light via an implanted optrode inhibits the activity of excitatory CA1 neurons. We found that light delivery to the hippocampus inhibited the recall of a contextual fear memory. Our results demonstrate that this optogenetic mouse line can be used to investigate the neuronal circuits underlying behavior.

Intragastric administration of glutamate increases REM sleep in rats.

Monosodium glutamate, a umami taste substance is commonly used flavor enhancer. The effect of intragastric administration of 1.5 ml of 0.12M monosodium glutamate on sleep-wake was studied in 10 adult male Wistar rats. Sleep-wake parameters were recorded through chronically implanted electroencephalogram, electrooculogram and electromyogram electrodes using a digital recording system (BIOPAC system Inc. BSL PRO 36, USA). The sleep-wake was recorded for 6h after the intragastric administration of either glutamate or saline. Sleep-wake stages were analyzed as wake, slow wave sleep and REM sleep. Compared to saline, intragastric administration of glutamate significantly increased REM sleep duration and episode frequency. REM sleep duration was increased in all the three 2h bins, 10:00-12:00 h (p=0.037), 12:00-14:00 h (p=0.037) and 14:00-16:00 h (p=0.007). The slow wave sleep and total sleep time were not affected. It is concluded that intragastric glutamate administration increases REM sleep.

Intracerebroventricular injection of orexin-2 receptor antagonist promotes REM sleep.

Orexins are important regulators of sleep-wakefulness (S-W). Rats were intracerebroventricularly (ICV) administered selective orexin-2 receptor antagonist, TCS-OX2-29. There was decreased wakefulness due to a decrease in the average wake episode duration and an increased REM sleep due to an increase in the number of REM episodes, indicating an inhibitory role of the central orexin-2 receptors on REM sleep generation.

Ambient temperature that induces maximum sleep in rats.

Changes in sleep and body temperature in rats at ambient temperatures below and above the self-selected temperature zone, are lacking in literature. In this report, the temperature preferred by the rats was first assessed before studying the changes in their sleep and body temperature, when they were exposed to ambient temperatures ranging from 18 degrees C to 36 degrees C. The rats preferred to stay at 27 degrees C when they were allowed to select their own ambient temperature, but maximum sleep was recorded when the rats were maintained at 30 degrees C. The ambient temperature-related changes in rapid eye movement sleep and deep slow wave sleep followed a bell-shaped curve, with a maximum at 30 degrees C. Of all the sleep parameters, rapid eye movement sleep showed a more marked ambient temperature-related change. Ambient temperatures above 33 degrees C and below 24 degrees C produced a significant reduction in sleep. Increase in sleep at 30 degrees C was associated with a slight elevation in body temperature, but a steep increase in body temperature at very high ambient temperature was associated with a decrease in sleep. Decrease in sleep at ambient temperatures below 24 degrees C was not related to any significant decrease in body temperature. Though maximum sleep was recorded at 30 degrees C, the sleep regulatory and thermoregulatory systems were at a functional equilibrium at 27 degrees C.