To sleep or not to sleep - Effects on memory in normal aging and disease.

Sleep behavior undergoes significant changes across the lifespan, and aging is associated with marked alterations in sleep amounts and quality. The primary sleep changes in healthy older adults include a shift in sleep timing, reduced slow-wave sleep, and impaired sleep maintenance. However, neurodegenerative and psychiatric disorders are more common among the elderly, which further worsen their sleep health. Irrespective of the cause, insufficient sleep adversely affects various bodily functions including energy metabolism, mood, and cognition. In this review, we will focus on the cognitive changes associated with inadequate sleep during normal aging and the underlying neural mechanisms.

Pontine control of rapid eye movement sleep and fear memory.

AIMS: We often experience dreams of strong irrational and negative emotional contents with postural muscle paralysis during rapid eye movement (REM) sleep, but how REM sleep is generated and its function remain unclear. In this study, we investigate whether the dorsal pontine sub-laterodorsal tegmental nucleus (SLD) is necessary and sufficient for REM sleep and whether REM sleep elimination alters fear memory. METHODS: To investigate whether activation of SLD neurons is sufficient for REM sleep induction, we expressed channelrhodopsin-2 (ChR2) in SLD neurons by bilaterally injecting AAV1-hSyn-ChR2-YFP in rats. We next selectively ablated either glutamatergic or GABAergic neurons from the SLD in mice in order to identify the neuronal subset crucial for REM sleep. We finally  investigated the role of REM sleep in consolidation of fear memory using rat model with complete SLD lesions. RESULTS: We demonstrate the sufficiency of the SLD for REM sleep by showing that photo-activation of ChR2 transfected SLD neurons selectively promotes transitions from non-REM (NREM) sleep to REM sleep in rats. Diphtheria toxin-A (DTA) induced lesions of the SLD in rats or specific deletion of SLD glutamatergic neurons but not GABAergic neurons in mice completely abolish REM sleep, demonstrating the necessity of SLD glutamatergic neurons for REM sleep. We then show that REM sleep elimination by SLD lesions in rats significantly enhances contextual and cued fear memory consolidation by 2.5 and 1.0 folds, respectively, for at least 9 months. Conversely, fear conditioning and fear memory trigger doubled amounts of REM sleep in the following night, and chemo-activation of SLD neurons projecting to the medial septum (MS) selectively enhances hippocampal theta activity in REM sleep; this stimulation immediately after fear acquisition reduces contextual and cued fear memory consolidation by 60% and 30%, respectively. CONCLUSION: SLD glutamatergic neurons generate REM sleep and REM sleep and SLD via the hippocampus particularly down-regulate contextual fear memory.

Glutamatergic pedunculopontine tegmental neurons control wakefulness and locomotion via distinct axonal projections.

STUDY OBJECTIVES: The pedunculopontine tegmental (PPT) nucleus is implicated in many brain functions, ranging from sleep/wake control and locomotion, to reward mechanisms and learning. The PPT contains cholinergic, GABAergic, and glutamatergic neurons with extensive ascending and descending axonal projections. Glutamatergic PPT (PPTvGlut2) neurons are thought to promote wakefulness, but the mechanisms through which this occurs are unknown. In addition, some researchers propose that PPTvGlut2 neurons promote locomotion, yet even though the PPT is a target for deep brain stimulation in Parkinson's disease, the role of the PPT in locomotion is debated. We hypothesized that PPTvGluT2 neurons drive arousal and specific waking behaviors via certain projections and modulate locomotion via others. METHODS: We mapped the axonal projections of PPTvGlut2 neurons using conditional anterograde tracing and then photostimulated PPTvGlut2 soma or their axon terminal fields across sleep/wake states and analyzed sleep/wake behavior, muscle activity, and locomotion in transgenic mice. RESULTS: We found that stimulation of PPTvGlut2 soma and their axon terminals rapidly triggered arousals from non-rapid eye movement sleep, especially with activation of terminals in the basal forebrain (BF) and lateral hypothalamus (LH). With photoactivation of PPTvGlut2 terminals in the BF and LH, this wakefulness was accompanied by locomotion and other active behaviors, but stimulation of PPTvGlut2 soma and terminals in the substantia nigra triggered only quiet wakefulness without locomotion. CONCLUSIONS: These findings demonstrate the importance of the PPTvGluT2 neurons in driving various aspects of arousal and show that heterogeneous brain nuclei, such as the PPT, can promote a variety of behaviors via distinct axonal projections.

Lateral hypothalamic neurotensin neurons promote arousal and hyperthermia.

Sleep and wakefulness are greatly influenced by various physiological and psychological factors, but the neuronal elements responsible for organizing sleep-wake behavior in response to these factors are largely unknown. In this study, we report that a subset of neurons in the lateral hypothalamic area (LH) expressing the neuropeptide neurotensin (Nts) is critical for orchestrating sleep-wake responses to acute psychological and physiological challenges or stressors. We show that selective activation of NtsLH neurons with chemogenetic or optogenetic methods elicits rapid transitions from non-rapid eye movement (NREM) sleep to wakefulness and produces sustained arousal, higher locomotor activity (LMA), and hyperthermia, which are commonly observed after acute stress exposure. On the other hand, selective chemogenetic inhibition of NtsLH neurons attenuates the arousal, LMA, and body temperature (Tb) responses to a psychological stress (a novel environment) and augments the responses to a physiological stress (fasting).

Ventrolateral periaqueductal gray mediates rapid eye movement sleep regulation by melanin-concentrating hormone neurons.

Neurons containing melanin-concentrating hormone (MCH) in the lateral hypothalamic area (LH) have been shown to promote rapid eye movement sleep (REMs) in mice. However, the downstream neural pathways through which MCH neurons influence REMs remained unclear. Because MCH neurons are considered to be primarily inhibitory, we hypothesized that these neurons inhibit the midbrain 'REMs-suppressing' region consisting of the ventrolateral periaqueductal gray and the lateral pontine tegmentum (vlPAG/LPT) to promote REMs. To test this hypothesis, we optogenetically inhibited MCH terminals in the vlPAG/LPT under baseline conditions as well as with simultaneous chemogenetic activation of MCH soma. We found that inhibition of MCH terminals in the vlPAG/LPT significantly reduced transitions into REMs during spontaneous sleep-wake cycles and prevented the increase in REMs transitions observed after chemogenetic activation of MCH neurons. These results strongly suggest that the vlPAG/LPT may be an essential relay through which MCH neurons modulate REMs.

Galanin neurons in the ventrolateral preoptic area promote sleep and heat loss in mice.

The preoptic area (POA) is necessary for sleep, but the fundamental POA circuits have remained elusive. Previous studies showed that galanin (GAL)- and GABA-producing neurons in the ventrolateral preoptic nucleus (VLPO) express cFos after periods of increased sleep and innervate key wake-promoting regions. Although lesions in this region can produce insomnia, high frequency photostimulation of the POAGAL neurons was shown to paradoxically cause waking, not sleep. Here we report that photostimulation of VLPOGAL neurons in mice promotes sleep with low frequency stimulation (1-4 Hz), but causes conduction block and waking at frequencies above 8 Hz. Further, optogenetic inhibition reduces sleep. Chemogenetic activation of VLPOGAL neurons confirms the increase in sleep, and also reduces body temperature. In addition, chemogenetic activation of VLPOGAL neurons induces short-latency sleep in an animal model of insomnia. Collectively, these findings establish a causal role of VLPOGAL neurons in both sleep induction and heat loss.

A Genetically Defined Circuit for Arousal from Sleep during Hypercapnia.

The precise neural circuitry that mediates arousal during sleep apnea is not known. We previously found that glutamatergic neurons in the external lateral parabrachial nucleus (PBel) play a critical role in arousal to elevated CO2 or hypoxia. Because many of the PBel neurons that respond to CO2 express calcitonin gene-related peptide (CGRP), we hypothesized that CGRP may provide a molecular identifier of the CO2 arousal circuit. Here, we report that selective chemogenetic and optogenetic activation of PBelCGRP neurons caused wakefulness, whereas optogenetic inhibition of PBelCGRP neurons prevented arousal to CO2, but not to an acoustic tone or shaking. Optogenetic inhibition of PBelCGRP terminals identified a network of forebrain sites under the control of a PBelCGRP switch that is necessary to arouse animals from hypercapnia. Our findings define a novel cellular target for interventions that may prevent sleep fragmentation and the attendant cardiovascular and cognitive consequences seen in obstructive sleep apnea. VIDEO ABSTRACT.

Cholinergic, Glutamatergic, and GABAergic Neurons of the Pedunculopontine Tegmental Nucleus Have Distinct Effects on Sleep/Wake Behavior in Mice.

The pedunculopontine tegmental (PPT) nucleus has long been implicated in the regulation of cortical activity and behavioral states, including rapid eye-movement (REM) sleep. For example, electrical stimulation of the PPT region during sleep leads to rapid awakening, whereas lesions of the PPT in cats reduce REM sleep. Though these effects have been linked with the activity of cholinergic PPT neurons, the PPT also includes intermingled glutamatergic and GABAergic cell populations, and the precise roles of cholinergic, glutamatergic, and GABAergic PPT cell groups in regulating cortical activity and behavioral state remain unknown. Using a chemogenetic approach in three Cre-driver mouse lines, we found that selective activation of glutamatergic PPT neurons induced prolonged cortical activation and behavioral wakefulness, whereas inhibition reduced wakefulness and increased non-REM (NREM) sleep. Activation of cholinergic PPT neurons suppressed lower-frequency electroencephalogram rhythms during NREM sleep. Last, activation of GABAergic PPT neurons slightly reduced REM sleep. These findings reveal that glutamatergic, cholinergic, and GABAergic PPT neurons differentially influence cortical activity and sleep/wake states. SIGNIFICANCE STATEMENT: More than 40 million Americans suffer from chronic sleep disruption, and the development of effective treatments requires a more detailed understanding of the neuronal mechanisms controlling sleep and arousal. The pedunculopontine tegmental (PPT) nucleus has long been considered a key site for regulating wakefulness and REM sleep. This is mainly because of the cholinergic neurons contained in the PPT nucleus. However, the PPT nucleus also contains glutamatergic and GABAergic neurons that likely contribute to the regulation of cortical activity and sleep-wake states. The chemogenetic experiments in the present study reveal that cholinergic, glutamatergic, and GABAergic PPT neurons each have distinct effects on sleep/wake behavior, improving our understanding of how the PPT nucleus regulates cortical activity and behavioral states.

Dynamic GABAergic afferent modulation of AgRP neurons.

Agouti-related peptide (AgRP) neurons of the arcuate nucleus of the hypothalamus (ARC) promote homeostatic feeding at times of caloric insufficiency, yet they are rapidly suppressed by food-related sensory cues before ingestion. Here we identify a highly selective inhibitory afferent to AgRP neurons that serves as a neural determinant of this rapid modulation. Specifically, GABAergic projections arising from the ventral compartment of the dorsomedial nucleus of the hypothalamus (vDMH) contribute to the preconsummatory modulation of ARCAgRP neurons. In a manner reciprocal to ARCAgRP neurons, ARC-projecting leptin receptor-expressing GABAergic vDMH neurons exhibit rapid activation upon availability of food that additionally reflects the relative value of the food. Thus, leptin receptor-expressing GABAergic vDMH neurons form part of the sensory network that relays real-time information about the nature and availability of food to dynamically modulate ARCAgRP neuron activity and feeding behavior.

Optogenetic-mediated release of histamine reveals distal and autoregulatory mechanisms for controlling arousal.

Histaminergic neurons in the tuberomammillary nucleus (TMN) are an important component of the ascending arousal system and may form part of a "flip-flop switch" hypothesized to regulate sleep and wakefulness. Anatomical studies have shown that the wake-active TMN and sleep-active ventrolateral preoptic nucleus (VLPO) are reciprocally connected, suggesting that each region can inhibit its counterpart when active. In this study, we determined how histamine affects the two branches of this circuit. We selectively expressed channelrhodopsin-2 (ChR2) in TMN neurons and used patch-clamp recordings in mouse brain slices to examine the effects of photo-evoked histamine release in the ventrolateral TMN and VLPO. Photostimulation decreased inhibitory GABAergic inputs to the ventrolateral TMN neurons but produced a membrane hyperpolarization and increased inhibitory synaptic input to the VLPO neurons. We found that in VLPO the response to histamine was indirect, most likely via a GABAergic interneuron. Our experiments demonstrate that release of histamine from TMN neurons can disinhibit the TMN and suppresses the activity of sleep-active VLPO neurons to promote TMN neuronal firing. This further supports the sleep-wake "flip-flop switch" hypothesis and a role for histamine in stabilizing the switch to favor wake states.

Human brain activity patterns beyond the isoelectric line of extreme deep coma.

The electroencephalogram (EEG) reflects brain electrical activity. A flat (isoelectric) EEG, which is usually recorded during very deep coma, is considered to be a turning point between a living brain and a deceased brain. Therefore the isoelectric EEG constitutes, together with evidence of irreversible structural brain damage, one of the criteria for the assessment of brain death. In this study we use EEG recordings for humans on the one hand, and on the other hand double simultaneous intracellular recordings in the cortex and hippocampus, combined with EEG, in cats. They serve to demonstrate that a novel brain phenomenon is observable in both humans and animals during coma that is deeper than the one reflected by the isoelectric EEG, and that this state is characterized by brain activity generated within the hippocampal formation. This new state was induced either by medication applied to postanoxic coma (in human) or by application of high doses of anesthesia (isoflurane in animals) leading to an EEG activity of quasi-rhythmic sharp waves which henceforth we propose to call ν-complexes (Nu-complexes). Using simultaneous intracellular recordings in vivo in the cortex and hippocampus (especially in the CA3 region) we demonstrate that ν-complexes arise in the hippocampus and are subsequently transmitted to the cortex. The genesis of a hippocampal ν-complex depends upon another hippocampal activity, known as ripple activity, which is not overtly detectable at the cortical level. Based on our observations, we propose a scenario of how self-oscillations in hippocampal neurons can lead to a whole brain phenomenon during coma.

Cellular mechanisms underlying EEG waveforms during coma.

This paper describes the various electroencephalographic (EEG) patterns expressed by the comatose brain, starting with the sleep-like oscillations associated with light coma. Deeper coma generally displays a burst-suppression pattern characterized by alternating episodes of isoelectric (flat) EEG and bursting slow waves. The latter are the result of cortical hyperexcitability, as demonstrated by intracellular recordings in anesthetized animals. Further deepening of the coma yields to continuous isoelectric EEG and eventually results in a newly discovered type of spiky waves that have been termed ν-complexes. They originate in the hippocampus as a result of intrinsically generated oscillations (ripples) in the delta range.

The hypocretins and their role in narcolepsy.

A series of discoveries spanning the last decade have uncovered a new neurotransmitter - hypocretin - and its role in energy metabolism, arousal, and addiction. Also, notably, a lack of hypocretin function has been unequivocally associated with the sleep disorder narcolepsy. Here we review these findings and discuss how they will influence future treatments of narcolepsy and other arousal and hyperarousal disorders. We introduce the concept of the hypocretin peptides and receptors and discuss the neuroanatomy and neurophysiology of the hypocretin system. A gain of function through pharmacolological and optogenetic means is also addressed in the following text, as is the loss of function: specifically narcolepsy in dogs, mice and humans and the challenges currently faced in treatment.

Cortical inhibition during burst suppression induced with isoflurane anesthesia.

Isoflurane is a widely used anesthetic which safely and reversibly induces deep coma and associated burst suppression (BS) electroencephalographic patterns. Here we investigate possible underlying causes for the state of cortical hyperexcitability which was recently shown to be one of the characteristics of BS. Our hypothesis was that cortical inhibition is diminished during isoflurane-induced BS. Experiments were performed in vivo using intracellular recordings of cortical neurons to assess their responsiveness to stimulations of connected thalamic nuclei. We demonstrate that during BS EPSPs were diminished by 44%, whereas inhibitory potentials were completely suppressed. This finding was supported by additional results indicating that a decrease in neuronal input resistance normally found during inhibitory responses under low isoflurane conditions was abolished in the BS condition. Moreover, removal of inhibition occasionally revealed excitatory components which were absent during recordings before the induction of BS. We also show that the absence of inhibition during BS is not caused by a blockage of GABA receptors, since iontophoretically applied GABA shows receptor availability. Moreover, the concentration of extracellular chloride was increased during BS, as would be expected after reduced flow of chloride through GABA(A) receptors. Also inhibitory responses were reinstated by selective blockage of glial glutamate transporters with dihydrokainate. These results suggest that the lack of inhibition during BS is caused by reduced excitation, probably resulting from increased glial uptake of glutamate stimulated by isoflurane, which creates a diminished activation of cortical interneurons. Thus cortical hyperexcitability during BS is favored by suppressed inhibition.

Hypersensitivity of the anesthesia-induced comatose brain.

Increasing levels of anesthesia are thought to produce a progressive loss of brain responsiveness to external stimuli. Here, we present the first report of a state window within anesthesia-induced coma, usually associated with an EEG pattern of burst suppression, during which brain excitability is dramatically increased so that even subliminal stimuli elicit bursts of whole-brain activity. We investigated this phenomenon in vivo using intracellular recordings of both neurons and glia, as well as extracellular calcium and EEG recordings. The results indicate that the bursting activity elicited with mechanical microstimulations, but also with auditory and visual stimuli, is dependent on complex mechanisms, including modulation of excitatory (NMDA) components, gap junction transmission, as well as the extracellular calcium concentration. The occurrence of bursting events is associated with a postburst refractory period that underlies the genesis of the alternating burst-suppression pattern. These findings raise the issue of what burst spontaneity during anesthesia-induced coma means and opens new venues for the handling of comatose patients.

Cholinergic action on cortical glial cells in vivo.

This study aims at understanding complex interactions between cortical neurons, glia and blood supply developing during the transition from slow-wave sleep to wakefulness. In spite of essential advances from in vitro and culture preparations, the basic mechanisms of glial interactions with their cellular and ionic environment had remained uninvestigated in vivo. Here we approach this issue by performing simultaneous intracellular recordings of cortical neurons and glia, together with measurements of cerebral blood flow (CBF), extracellular K+ concentrations and local field potentials in both anesthetized (ketamine-xylazine) and naturally behaving cats. Under anesthesia, cortical activation was elicited with electric stimulation of cholinergic nuclei (pedunculopontine tegmental in the brainstem and/or nucleus basalis in the basal forebrain). Iontophoretic application of acetylcholine on the recorded cells was also used. In the vast majority of cases (> 80%) glial cells were hyperpolarized during electric stimulation or spontaneous activation. This result was also obtained in all cases where iontophoresis was used or when glutamatergic kainate/quisqualate receptors were blocked with 6-cyano-7-nitroquinoxaline-2,3-dione. The glial hyperpolarization was associated with steady neuronal depolarization, increased CBF, lower extracellular K+ concentration, increased membrane resistance, decreased membrane capacitance and persistent positive DC field potentials. In some cases of cortical activation (< 20%), glial cells displayed sustained depolarizing potentials, in parallel with neuronal depolarization, decreased CBF and more negative DC field potentials. The above-mentioned effects of cholinergic activation were blocked by the muscarinic antagonist scopolamine. We propose that the glial response to cholinergic activation results from the balance between the direct hyperpolarizing action of acetylcholine and the depolarizing modulation of glutamate from the neighboring neurons, in addition to the modulation of the interglial communication pathway and/or the ionic traffic across blood vessels.