- 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.

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

RESULT

· Activation of LH_GABA 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.

The authors mapped LH_GABA 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 LH_GABA 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 LH_GABA terminals in the TRN elicited the fastest sleep-to-wake transitions.

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.

· 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 LH_GABA cells resulted in prolonged NREM sleep, as seen by slower, more pronounced delta waves.

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

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 LH_GABA 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 LH_GABA 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. 

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]).