Searching for sleep in all the right places: My career in sleep research.

My research has always focused on sleep, whether monitoring neural activity (microwires, c-Fos, calcium imaging), triggering it with optogenetics or pharmacologically (anandamide, cholinergic agonists), or measuring levels of endogenous sleep agents such as adenosine. A recurring theme of my research is to use new tools to find the sweet spot in the brain where the signal to sleep begins. My goal is to identify the circuit, determine whether it degrades with age or disease, and repair the circuit when it fails.

Role of the Locus Coeruleus Arousal Promoting Neurons in Maintaining Brain Criticality across the Sleep-Wake Cycle.

Sleep control depends on a delicate interplay among brain regions. This generates a complex temporal architecture with numerous sleep-stage transitions and intermittent fluctuations to micro-states and brief arousals. These temporal dynamics exhibit hallmarks of criticality, suggesting that tuning to criticality is essential for spontaneous sleep-stage and arousal transitions.

Most dynorphin neurons in the zona incerta-perifornical area are active in waking relative to non-rapid-eye movement and rapid-eye movement sleep.

Dynorphin is an endogenous opiate localized in many brain regions and spinal cord, but the activity of dynorphin neurons during sleep is unknown. Dynorphin is an inhibitory neuropeptide that is coreleased with orexin, an excitatory neuropeptide. We used microendoscopy to test the hypothesis that, like orexin, the dynorphin neurons are wake-active.

Unilateral optogenetic stimulation of Lhx6 neurons in the zona incerta increases REM sleep.

To determine how a waking brain falls asleep researchers have monitored and manipulated activity of neurons and glia in various brain regions. While imaging Gamma-Aminobutyric Acid (GABA) neurons in the zona incerta (ZI) we found a subgroup that anticipates onset of NREM sleep (Blanco-Centurion C, Luo S, Vidal-Ortiz A, Swank C, Shiromani PJ. Activity of a subset of vesicular GABA-transporter neurons in the ventral ZI anticipates sleep onset.

Activity of GABA neurons in the zona incerta and ventral lateral periaqueductal grey is biased towards sleep.

Study Objectives: As in various brain regions the activity of gamma-aminobutyric acid (GABA) neurons is largely unknown, we measured in vivo changes in calcium fluorescence in GABA neurons in the zona incerta (ZI) and the ventral lateral periaqueductal grey (vlPAG), two areas that have been implicated in regulating sleep.

Methods: vGAT-Cre mice were implanted with sleep electrodes, microinjected with rAAV-DIO-GCaMP6 into the ZI (n = 6) or vlPAG (n = 5) (isoflurane anesthesia) and a GRIN (Gradient-Index) lens inserted atop the injection site. Twenty-one days later, fluorescence in individual vGAT neurons was recorded over multiple REM cycles.

Mapping Network Activity in Sleep.

It was in the influenza pandemic of 1918 that von Economo identified specific brain regions regulating sleep and wake. Since then researchers have used a variety of tools to determine how the brain shifts between states of consciousness. In every enterprise new tools have validated existing data, corrected errors and made new discoveries to advance science.

Activity of a subset of vesicular GABA-transporter neurons in the ventral zona incerta anticipates sleep onset.

Study Objectives: Sleep and wake are opposing behavioral states controlled by the activity of specific neurons that need to be located and mapped. To better understand how a waking brain falls asleep it is necessary to identify activity of individual phenotype-specific neurons, especially neurons that anticipate sleep onset. In freely behaving mice, we used microendoscopy to monitor calcium (Ca2+) fluorescence in individual hypothalamic neurons expressing the vesicular GABA transporter (vGAT), a validated marker of GABA neurons.

Amygdala GABA Neurons Project To vlPAG And mPFC.

The amygdala regulates multiple behaviors and emotions by projecting to multiple brain regions. However, the topographical distribution of amygdala neurons projecting to specific brain areas is still unclear. In the present study, we focus on determining whether single amygdala neurons project to the brain stem ventrolateral periaqueductal grey (vlPAG) and to the medial prefrontal cortex (mPFC).

Dynamic Network Activation of Hypothalamic MCH Neurons in REM Sleep and Exploratory Behavior.

Most brain neurons are active in waking, but hypothalamic neurons that synthesize the neuropeptide melanin-concentrating hormone (MCH) are claimed to be active only during sleep, particularly rapid eye movement (REM) sleep. Here we use deep-brain imaging to identify changes in fluorescence of the genetically encoded calcium (Ca) indicator GCaMP6 in individual hypothalamic neurons that contain MCH. An electrophysiology study determined a strong relationship between depolarization and Ca fluorescence in MCH neurons.

VGAT and VGLUT2 expression in MCH and orexin neurons in double transgenic reporter mice.

The neuropeptides orexin and melanin-concentrating hormone (MCH), as well as the neurotransmitters GABA (γ-Aminobutyric acid) and glutamate are chief modulators of the sleep-wake states in the posterior hypothalamus. To investigate co-expression of vesicular GABA transporter (VGAT, a marker of GABA neurons) and the vesicular glutamate transporter-2 (VGLUT2, a marker of glutamate neurons) in orexin and MCH neurons, we generated two transgenic mouse lines. One line selectively expressed the reporter gene EYFP in VGAT+ neurons, whereas the other line expressed reporter gene tdTomato in VGLUT2+ neurons.

Rewiring brain circuits to block cataplexy in murine models of narcolepsy.

Narcolepsy was first identified almost 130 years ago, but it was only 15 years ago that it was identified as a neurodegenerative disease linked to a loss of orexin neurons in the brain. It is unclear what causes the orexin neurons to die, but our strategy has been to place the gene for orexin into surrogate neurons in the validated mouse models of narcolepsy, and test whether it can block narcolepsy symptoms, such as cataplexy. In both the orexin knockout and the orexin-ataxin-3 mouse models of narcolepsy we have found that cataplexy can be blocked if the surrogate neurons are part of the circuit responsible for cataplexy.

New Neuroscience Tools That Are Identifying the Sleep-Wake Circuit.

The complexity of the brain is yielding to technology. In the area of sleep neurobiology, conventional neuroscience tools such as lesions, cell recordings, c-Fos, and axon-tracing methodologies have been instrumental in identifying the complex and intermingled populations of sleep- and arousal-promoting neurons that orchestrate and generate wakefulness, NREM, and REM sleep. In the last decade, new technologies such as optogenetics, chemogenetics, and the CRISPR-Cas system have begun to transform how biologists understand the finer details associated with sleep-wake regulation.

Optogenetic activation of melanin-concentrating hormone neurons increases non-rapid eye movement and rapid eye movement sleep during the night in rats.

Neurons containing melanin-concentrating hormone (MCH) are located in the hypothalamus. In mice, optogenetic activation of the MCH neurons induces both non-rapid eye movement (NREM) and rapid eye movement (REM) sleep at night, the normal wake-active period for nocturnal rodents [R. R.

Orexin gene transfer into the amygdala suppresses both spontaneous and emotion-induced cataplexy in orexin-knockout mice.

Narcolepsy is a chronic sleep disorder linked to the loss of orexin-producing neurons in the hypothalamus. Cataplexy, a sudden loss of muscle tone during waking, is an important distinguishing symptom of narcolepsy and it is often triggered by strong emotions. The neural circuit underlying cataplexy attacks is not known, but is likely to involve the amygdala, a region implicated in regulating emotions.

Optogenetic stimulation of astrocytes in the posterior hypothalamus increases sleep at night in C57BL/6J mice.

A distributed network of neurons regulates wake, non-rapid eye movement (NREM) sleep, and REM sleep. However, there are also glia in the brain, and there is growing evidence that neurons and astroglia communicate intimately to regulate behaviour. To identify the effect of optogenetic stimulation of astrocytes on sleep, the promoter for the astrocyte-specific cytoskeletal protein, glial fibrillary acidic protein (GFAP) was used to direct the expression of channelrhodopsin-2 (ChR2) and the linked reporter gene, enhanced yellow fluorescent protein (EYFP), in astrocytes.

Neurons containing orexin or melanin concentrating hormone reciprocally regulate wake and sleep.

Neurons containing orexin (hypocretin), or melanin concentrating hormone (MCH) are intermingled with each other in the perifornical and lateral hypothalamus. Each is a separate and distinct neuronal population, but they project to similar target areas in the brain. Orexin has been implicated in regulating arousal since loss of orexin neurons is associated with the sleep disorder narcolepsy.

Endocannabinoid modulation of cortical up-states and NREM sleep.

Up-/down-state transitions are a form of network activity observed when sensory input into the cortex is diminished such as during non-REM sleep. Up-states emerge from coordinated signaling between glutamatergic and GABAergic synapses and are modulated by systems that affect the balance between inhibition and excitation. We hypothesized that the endocannabinoid (EC) system, a neuromodulatory system intrinsic to the cortical microcircuitry, is an important regulator of up-states and sleep.

Optogenetic stimulation of MCH neurons increases sleep.

Melanin concentrating hormone (MCH) is a cyclic neuropeptide present in the hypothalamus of all vertebrates. MCH is implicated in a number of behaviors but direct evidence is lacking. To selectively stimulate the MCH neurons the gene for the light-sensitive cation channel, channelrhodopsin-2, was inserted into the MCH neurons of wild-type mice.

Effects of orexin gene transfer in the dorsolateral pons in orexin knockout mice.

Study Objectives: Narcolepsy is a sleep disorder characterized by loss of orexin neurons. Previously, our group demonstrated that transfer of the orexin gene into surrogate neurons in the lateral hypothalamus and the zona incerta significantly reduced cataplexy bouts in the orexin-ataxin-3 mice model of narcolepsy. The current study determined the effects of orexin gene transfer into the dorsolateral pontine neurons in the orexin knockout (KO) mice model of narcolepsy.

Orexin gene transfer into zona incerta neurons suppresses muscle paralysis in narcoleptic mice.

Cataplexy, a sudden unexpected muscle paralysis, is a debilitating symptom of the neurodegenerative sleep disorder, narcolepsy. During these attacks, the person is paralyzed, but fully conscious and aware of their surroundings. To identify potential neurons that might serve as surrogate orexin neurons to suppress such attacks, the gene for orexin (hypocretin), a peptide lost in most human narcoleptics, was delivered into the brains of the orexin-ataxin-3 transgenic mouse model of human narcolepsy.

Hypocretin-2 saporin lesions of the ventrolateral periaquaductal gray (vlPAG) increase REM sleep in hypocretin knockout mice.

Ten years ago the sleep disorder narcolepsy was linked to the neuropeptide hypocretin (HCRT), also known as orexin. This disorder is characterized by excessive day time sleepiness, inappropriate triggering of rapid-eye movement (REM) sleep and cataplexy, which is a sudden loss of muscle tone during waking. It is still not known how HCRT regulates REM sleep or muscle tone since HCRT neurons are localized only in the lateral hypothalamus while REM sleep and muscle atonia are generated from the brainstem.

Activity of pontine neurons during sleep and cataplexy in hypocretin knock-out mice.

Narcolepsy is a human sleep disorder resulting from the loss of neurons containing the neuropeptide orexin, also known as hypocretin. Cataplexy, which is a sudden loss of muscle tone during waking, is an important diagnostic symptom of narcolepsy. In humans and canines with narcolepsy, cataplexy is considered to be a separate and distinct behavioral state.