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Brain Basics - An Introduction to Deep Sleep

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Brain Basics - An Introduction to Deep Sleep

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Deep sleep (i.e. NREM sleep) is essential for our health and wellbeing; however, the neural mechanisms that underlie deep sleep remain incompletely understood. Studying the neurobiology of sleep may reveal important insights into sleep-related pathologies and novel sleep-improvement technology.

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The brain benefits of deep sleep -- and how to get more of it |  Dan Gartenberg

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Sleep is divided into NREM and REM sleep, which are characterized by differences in EEG patterns. [Lee, 2012]

NREM: (stages 1-4) progressively increased synchronicity in brain wave activity.
--> high amplitude, low frequency waves

REM: unsynchronized brain wave activity, similar to the wakefulness state.
--> low amplitude, high frequency waves

Different neural mechanisms govern NREM and REM sleep.

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DigiCortex simulation of the thalamocortical system with 16.7 million neurons.

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- Corticothalamic (CT) cells send excitatory projections to thalamocortical (TC) cells within the thalamus, which send excitatory projections back to the cortex.  CT and TC cells coordinate slow-wave oscillations. [Steriade, 1993]

- Thalamic reticular nucleus (TRN) provides inhibitory input to TC cells. Active TRN cells generate spindles and slow waves (hallmarks of NREM sleep!) [Kim, 2012]

- Inhibitory post-synaptic currents (IPSCs) have been recorded in TRN cells in vitro. [Schofield, 2007] The TRN has been shown to be modulated by extra-thalamic GABA input, but no study has shown the origin of these inhibitory inputs!

Image: Original artwork.

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- GABA cells within the lateral hypothalamus (LH) are known to control sleep and awake processing (i.e. arousal, attention, reward, and stress). [Kempadoo, 2013]

- LH is a good candidate to be an inhibitory modulator of the TRN!

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GABAergic neurons within the lateral hypothalamus (LHGABA) inhibit TRN cells, thereby altering thalamocortical activity to induce arousal during NREM sleep.

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Channelrhodpsin:
- Blue light activated
- Na⁺ Cation influx induces depolarization in neurons
Archaerhodopsin:
- Yellow/orange light activated
- Hydrogen cations efflux induces hyperpolarization in neurons

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Optogenetic activation of Lateral Hypothalamus GABAergic terminals  in the TRN induces rapid awakening from NREM, but not REM

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Optogenetic activation of Lateral Hypothalamus GABAergic terminals  in the TRN induces rapid awakening from NREM, but not REM

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METHOD
The authors injected Cre-inducible AAVdj vectors containing ChETA-eYFP into the LH of Tg (VGAT-Cre) mice (transgene mice with Cre recombinase under a VGAT promoter). Optogenetic activation was done 10 seconds after NREM sleep onset for a duration of 10 seconds. Light-dependent activation was done at 1 Hz, 20 Hz and 1 second continuous

RESULT

· Activation of LHGABA during NREM sleep caused sudden sleep-to-wake transitions (in less than 3 seconds). Notably, there was no change observed when mice were in REM sleep.

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Various regions involved in sleep regulation receive monosynaptic inputs from LH GABAergic neurons

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Various regions involved in sleep regulation receive monosynaptic inputs from LH GABAergic neurons

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METHOD

The authors mapped LHGABA terminals in other areas of the brain using a wide variety of different methods: ChR2 assisted circuit mapping, anterograde tracing, retrograde tracing (#1), retrograde tracing (#2) and optogenetic stimulation.

RESULT

· Activation of LHGABA terminals in the TRN elicited similar results as previously shown (rapid arousal only during NREM sleep), however activation of other areas (like the locus coeruleus, medial septum and paraventricular fibers) also elicited sleep-to-wake transitions during both NREM and REM. Notably, activation of LHGABA terminals in the TRN elicited the fastest sleep-to-wake transitions.

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- During NREM, sleep-to-wake transitions, optogenetic activation of LHGABA  cells exert a strong inhibition on TRN cells, which progressively result in a dis-inhibition of thalamocortical (TC) cells.

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1. Good use of alternate methods to validate findings. Such as retrograde and anterograde tracing using fluorescent beads, GFP lentivirus, and ChETA-YFP when mapping parts of the brain. This strengthens their claim of a TRN-LH connection as it is less likely to be an artifact if their observations remain the same across multiple experimental methods.

2. The authors activated and inhibited neurons in the brain regions of interest which supported their hypothesis in multiple conditions.

3.  Herrera et al. is the first group to show that the TRN receives inputs from LHGABA neurons.

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Hypothesis 1:
Do the LC and/or the PVf interact with the thalamocortical network?
Herrera et al. found inducing LHGABA neurons connected to the LC and PVf also resulted in arousal from NREM sleep (as well as REM sleep for the LC). This suggests other possible mechanisms or a more complex mechanism in sleep-to-wake transitions. If the LC and PVf do interact with the thalamocortical network, that would suggest the pathway is the sole pathway responsible for sleep-to-wake transitions. However, it is also possible the LC and PVf work on other regions of the brain to induce wakefulness and would be value to elucidate for future sleep research.

Experiment:
1. Inject LC or PVf cell bodies with red fluorescent beads.
2. Inject the CT, TC, or TRN with GFP lentivirus.
3. Harvest mouse brain.
4. Cryosection brain slices.
5. Epifluorescent microscopy of neuron synapses.

If the LC/PVf interact with the thalamocortical network one would expect to see synapses formed between the LC or PVf and the CT, TC, or TRN. If our resulsts did not show connections with the thalamocortical network, it's possible the LC or PVf interact with orexin neurons in the LH which have been shown to be involved in sleep to wake transitions as well [de Lecea, 2014]. To address this, one could Co-stain sections for hypocretin-1 (orexin neuron marker) [Baumann 2010] to visualize connections with these neurons.

Image:  Original artwork.

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Optogenetic silencing of Lateral Hypothalamus GABAergic terminals in the TRN induces prolonged NREM sleep

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Optogenetic silencing of Lateral Hypothalamus GABAergic terminals in the TRN induces prolonged NREM sleep

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METHOD

· The authors injected Cre-inducible AAVdj vectors containing ArchT-eYFP into the LH of Tg (VGAT-Cre) mice. Optogenetic silencing was done 10 seconds after NREM sleep onset for a duration of 10 seconds. Light-dependent activation was done at 1 Hz, 20 Hz and 1 second continuous

RESULT

· Optogenetic silencing of LHGABA cells resulted in prolonged NREM sleep, as seen by slower, more pronounced delta waves.

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Ed Boyden discusses the use of optogenetics in neuronal activation and silencing

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Ed Boyden discusses the use of optogenetics in neuronal activation and silencing

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Massachusetts Institute of Technology (MIT)

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Papers:

Barone, F.C., Cheng, J.T. & Wayner, M.J. Reticular thalamic inhibitory input to lateral hypothalamic neurons: a functional and histochemical determination. Brain Res. Bull. 1994; 33, 575–582.

Baumann, C. R. et al. Loss of hypocretin (orexin) neurons with traumatic brain injury. Ann. Neurol. 66, 555–559 (2009).

Castle MJ, Turunen HT, Vandenberghe LH, Wolfe JH. Controlling AAV Tropism in the Nervous System with Natural and Engineered Capsids. Methods Mol Biol. 2016;1382:133-49.

de Lecea, L. & Huerta, R. Hypocretin (orexin) regulation of sleep-to-wake transitions. Front. Pharmacol. 5, 16 (2014).

Goncalves, S.B.; Palha, J.M.; Fernandes, H.C.; Souto, M.R.; Pimenta, S.; Dong, T.; Yang, Z.; Ribeiro, J.F.; Correia, J.H. LED Optrode with Integrated Temperature Sensing for Optogenetics. Micromachines 2018, 9, 473.

Herrera, C. G. et al. Hypothalamic feedforward inhibition of thalamocortical network controls arousal and consciousness. Nat. Neurosci. 19, 290 (2015).

Jego, S. et al. Optogenetic identification of a rapid eye movement sleep modulatory circuit in the hypothalamus. Nat. Neurosci. 16, 1637–1643 (2013).

Kandratavicius, L. et al. Animal models of epilepsy: use and limitations. Neuropsychiatr. Dis. Treat. 10, 1693–1705 (2014).

Kempadoo, K.A. et al. Hypothalamic neurotensin projections promote reward by enhancing glutamate transmission in the VTA. J. Neurosci. 2013; 33, 7618–7626.

Kim, A. et al. Optogenetically induced sleep spindle rhythms alter sleep architectures in mice. Proc. Natl. Acad. Sci. USA. 2012; 109, 20673–20678.

Lee, S.-H. & Dan, Y. Neuromodulation of brain states. Neuron. 2012; 76: 209–222.

Lin, J. Y. A user’s guide to channelrhodopsin variants: features, limitations and future developments. Exp. Physiol. 96, 19–25 (2011).

McCormick, D.A. & Bal, T. Sleep and arousal: thalamocortical mechanisms. Annu. Rev. Neurosci. 1997; 20, 185–215.

Paz, J. T. & Huguenard, J. R. Optogenetics and epilepsy: past, present and future. Epilepsy Curr. 15, 34–38 (2015).

Qin, J. Y. et al. Systematic Comparison of Constitutive Promoters and the Doxycycline-Inducible Promoter. PLoS One 5, e10611 (2010).

San Gil, R., Ooi, L., Yerbury, J. J. & Ecroyd, H. The heat shock response in neurons and astroglia and its role in neurodegenerative diseases. Mol. Neurodegener. 12, 65 (2017).

Schofield, C.M. & Huguenard, J.R. GABA affinity shapes IPSCs in thalamic nuclei. J. Neurosci. 2007; 27: 7954–7962.

Steriade M et al. A novel slow (<1 Hz) oscillation of neocortical neurons in vivo: depolarizing and hyperpolarizing components. J Neurosci. 13: 3252-3265.

Rordorf, G., Koroshetz, W. J. & Bonventre, J. V. Heat shock protects cultured neurons from glutamate toxicity. Neuron 7, 1043–1051 (1991).

Images & Videos:
1) https://youtu.be/1U2qMRGihGg
2) https://giphy.com/gifs/cells-2GOBR8oGvKBG
3) https://www.youtube.com/watch?v=FhosaQSWCFw&t=1s
4) https://www.youtube.com/watch?v=Nb07TLkJ3Ww
5https://www.kisspng.com/png-incandescent-light-bulb-brain-lamp-clip-art-light-1278429/
6)https://www.hhmi.org/news/new-platform-brain-wide-imaging-and-reconstruction-neurons
7) https://leader.pubs.asha.org/article.aspx?articleid=1921132

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Hypothesis 2:
Is the LH involved in the pathophysiology of epilepsy?

Hyper-activation of the thalamocortical network has been associated with some epilepsies [Barone, 1994]. Herrera et al. only activated the LH for a maximum of 10 seconds, a longer activation could result in thalamocortical network over-activation. If the LH can modulate the thalamocortical network, and the thalamocortical network is sometimes overactive in seizures [Barone, 1994], the LH could be a novel target in seizures management. This would also fit well with current objectives using optogenetics as a possible seizure therapeutic [Paz, 2015].

Experiment:
1. Inject the LH with AAVdj-ChETA-YFP (same protocol as used in this study).
2. Over-stimulate the LH at various levels.
3. Collect EEG data to assess seizure activity.

If the LH does control the thalamocortical network from becoming overactive, overstimulating LHGABA neurons for a long period of time may induce seizure activity. Alternatively, it may be valuable to induce seizure activity in mice (eg. by chemoconvulsants [Kandratavicius, 2014]) and observe seizure recovery between LHGABA overactive vs controls to assess whether the LH has a role in restoring normal function during a seizure event.
Seizures are a symptom that arise from a highly heterogeneous group of neurological disorders. If these experiments do not show an affect on seizure activity this does not necessarily mean that the LH has no role in seizure modulation, only that its disregulation does not induce seizures or cannot recover chemoconvulsant induced seizures.

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1. No validation of off-target rhodopsin activation. Their only control for cell specific expression was GABAergic specific expression, and would be expressed in many cells throughout the brain. The authors did not validate that activation was localized specifically to the region of interest.

2. Some have suggested that AAV2 better suited for targeting small regions in the brain as the AAVdj used in this study has such a high tropism for neurons that it likely infected cells outside of their region of interest [Castle, 2016] - making off target rhodopsin activation is more likely.

3. The EF1a promoter used to control the rhodopsin transgene is a strong constitutive promoter [Qin, 2010]. This would result in highly overexposed rhodopsin, which has been critiqued for disrupting the native membrane environment [Lin, 2011].  Moreover, this was not controlled for with their YFP control group as the YFP was not membrane bound.

4. Used continuous light activation which has been shown to lead to overheating in brain tissue [Goncalves, 2018]. This would be more acceptable if the authors had validated that the brain tissue was not damaged (eg. using an optogenetic probe with a built in thermometer [Goncalves, 2018] or assessing neuron heat shock response with HPS70 [Rordorf 1991, San Gil 2017]).

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Major Findings
1. Optogenetic activation of LHGABA  neurons induced arousal from NREM sleep.
2. Optogenetic silencing of LHGABA  neurons did not induce arousal from NREM sleep.
These results align with the hypothesis that LHGABA  cells act as an inhibitory modulator of the TRN, and thereby alter thalamocortical activity to induce arousal from NREM sleep.

Novelty and Impact
Previous reports have elucidated the role of the thalamocortical system within NREM sleep, however, this study was the first to identify a source of GABAergic input onto the TRN.

Previous reports also have demonstrated that optogenetic activation of hypocretin neurons within the lateral hypothalamus (LHhcrt)  and noradrenergic neurons within the locus coeruleus (LCNE)  have induced wakefulness from NREM and REM sleep. [Jego, 2013
- The results from this study showed that wakefulness induced by the LH-TRN circuit was faster than wakefulness induced by LHhcrt and  LCNE  neurons.
- This suggests that there is a high degree of specialization within arousal circuits. Further investigation is required fully elucidate this mechanism.

The results of this study indicated that transient activation of LHGABA  neurons induce a sleep-to-wake transition from NREM sleep. Since LH cells are only active for a short period of time, this suggests that these cells receive inhibitory input. A previous study found that the TRN reciprocally inactivates LHGABA  neurons [Barone, 1994]. This corroborates the relationship between the LH and TRN.