Most dynorphin neurons in the zona incerta-perifornical area are active in waking relative to NREM and REM 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 use microendoscopy to test the hypothesis that, like orexin, the dynorphin neurons are wake-active. Dynorphin-cre mice (n=3) were administered rAAV8-Ef1a-Con/Foff 2.0-GCaMP6M into the zona incerta-perifornical area, implanted with a GRIN lens (Gradient Reflective Index), and electrodes to the skull recorded sleep. One month later, a miniscope imaged calcium fluorescence in dynorphin neurons during multiple bouts of wake, NREM, and REM sleep. Unbiased data analysis identified changes in calcium fluorescence in sixty-four dynorphin neurons. Most of the dynorphin neurons (72%) had the highest fluorescence during bouts of active and quiet waking compared to NREM or REM sleep; a subset (20%) were REM-max. Our results are consistent with the emerging evidence that the activity of orexin neurons can be classified as wake-max or REM-max. Since the two neuropeptides are coexpressed and coreleased, we suggest that dynorphin-cre-driven calcium sensors could increase understanding of the role of this endogenous opiate in pain and sleep.

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 GABA neurons in the zona incerta (ZI) we found a subgroup that anticipates onset of NREM sleep 1. To differentiate the GABA subtype we now image and optogenetically manipulate the ZI neurons containing the transcription factor, Lhx6. In the first study Lhx6-cre mice (n=5; female=4) were given rAAV-DJ-EF1a-DIO-GCaMP6M into the ZI (isofluorane anesthesia), a GRIN lens implanted, and 21d later sleep and fluorescence in individual Lhx6 neurons were recorded for 4h. Calcium fluorescence was detected in 132 neurons. 45.5% of the Lhx6 neurons were REM-max; 30.3% were Wake-max; 11.4% were wake+REM max; 9% were NREM-max; and 3.8% had no change. The NREM-max group of neurons fluoresced 30s ahead of sleep onset. The second study tested the effects of unilateral optogenetic stimulation of the ZI Lhx6 neurons (n=14 mice) (AAV5-Syn-FLEX-rc[ChrimsonR-tdTomato]. Stimulation at 1 and 5Hz (1 minute on- 4 minute off) significantly increased percent REM sleep during the 4h stimulation period (last half of day cycle). The typical experimental approach is to stimulate neurons in both hemispheres, but here we found that low frequency stimulation of ZI Lhx6 neurons in one hemisphere is sufficient to shift states of consciousness. Detailed mapping combined with mechanistic testing is necessary to identify local nodes that can shift the brain between wake-sleep states.

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. Regions of interest corresponding to individual vGAT somata were automatically extracted with PCA-ICA analysis. RESULTS: In the ZI, 372 neurons were identified. Previously, we had recorded the activity of 310 vGAT neurons in the ZI and we combined the published dataset with the new dataset to create a comprehensive dataset of ZI vGAT neurons (total neurons = 682; mice = 11). In the vlPAG, 169 neurons (mice = 5) were identified. In both regions, most neurons were maximally active in REM sleep (R-Max; ZI = 51.0%, vlPAG = 60.9%). The second most abundant group was W-Max (ZI = 23.9%, vlPAG = 25.4%). In the ZI, but not in vlPAG, there were neurons that were NREMS-Max (11.7%). vlPAG had REMS-Off neurons (8.3%). In both areas, there were two minor classes: wake/REMS-Max and state indifferent. In the ZI, the NREMS-Max neurons fluoresced 30 s ahead of sleep onset. CONCLUSIONS: These descriptive data show that the activity of GABA neurons is biased in favor of sleep in two brain regions implicated in sleep.

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. The brain is a challenge but new tools can disentangle the brain network. We summarize the newest tool, a miniature microscope, that provides unprecedented view of activity of glia and neurons in freely behaving mice. With this tool we have observed that the activity of a majority of GABA and MCH neurons in the lateral hypothalamus is heavily biased toward sleep. We suggest that miniscope data identifies activity at the cellular level in normal versus diseased brains, and also in response to specific hypnotics. Shifts in activity in small networks across the brain will help identify point of criticality that switches the brain from wake to sleep.

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. METHODS: vGAT-Cre mice (male = 3; female = 2) transfected with rAAV-FLEX-GCaMP6M in the lateral hypothalamus were imaged 30 days later during multiple episodes of waking (W), non-rapid-eye movement sleep (NREMS) or REMS (REMS). RESULTS: 372 vGAT neurons were recorded in the zona incerta. 23.9% of the vGAT neurons showed maximal fluorescence during wake (classified as wake-max), 4% were NREM-max, 56.2% REM-max, 5.9% wake/REM max, while 9.9% were state-indifferent. In the NREM-max group, Ca2+ fluorescence began to increase before onset of NREM sleep, remained high throughout NREM sleep, and declined in REM sleep. CONCLUSIONS: We found that 60.2% of the vGAT GABA neurons in the zona incerta had activity that was biased towards sleep (NREM and REMS). A subset of vGAT neurons (NREM-max) became active in advance of sleep onset and may induce sleep by inhibiting the activity of the arousal neurons. Abnormal activation of the NREM-max neurons may drive sleep attacks and hypersomnia.

Activity dynamics of amygdala GABAergic neurons during cataplexy of narcolepsy.

Recent studies showed activation of the GABAergic neurons in the central nucleus of the amygdala (CeA) triggered cataplexy of sleep disorder narcolepsy. However, there is still no direct evidence on CeA GABAergic neurons' real-time dynamic during cataplexy. We used a deep brain calcium imaging tool to image the intrinsic calcium transient as a marker of neuronal activity changes in the narcoleptic VGAT-Cre mice by expressing the calcium sensor GCaMP6 into genetically defined CeA GABAergic neurons. Two distinct GABAergic neuronal groups involved in cataplexy were identified: spontaneous cataplexy-ON and predator odor-induced cataplexy-ON neurons. Majority in the latter group were inactive during regular sleep/wake cycles but were specifically activated by predator odor and continued their intense activities into succeeding cataplexy bouts. Furthermore, we found that CeA GABAergic neurons became highly synchronized during predator odor-induced cataplexy. We suggest that the abnormal activation and synchronization of CeA GABAergic neurons may trigger emotion-induced cataplexy.

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 (Ca2+) indicator GCaMP6 in individual hypothalamic neurons that contain MCH. An in vitro electrophysiology study determined a strong relationship between depolarization and Ca2+ fluorescence in MCH neurons. In 10 freely behaving MCH-cre mice (male and female), the highest fluorescence occurred in all recorded neurons (n = 106) in REM sleep relative to quiet waking or non-REM sleep. Unexpectedly, 70% of the MCH neurons had strong fluorescence activity when the mice explored novel objects. Spatial and temporal mapping of the change in fluorescence between pairs of MCH neurons revealed dynamic activation of MCH neurons during REM sleep and activation of a subset of the same neurons during exploratory behavior. Functional network activity maps will facilitate comparisons of not only single-neuron activity, but also network responses in different conditions and disease.SIGNIFICANCE STATEMENT Functional activity maps identify brain circuits responding to specific behaviors, including rapid eye movement sleep (REM sleep), a sleep phase when the brain is as active as in waking. To provide the first activity map of individual neurons during REM sleep, we use deep-brain calcium imaging in unrestrained mice to map the activity of hypothalamic melanin-concentrating hormone (MCH) neurons. MCH neurons were found to be synchronously active during REM sleep, and also during the exploration of novel objects. Spatial mapping revealed dynamic network activation during REM sleep and activation of a subset of the neurons during exploratory behavior. Functional activity maps at the cellular level in specific behaviors, including sleep, are needed to establish a brain connectome.