L5 - Hypothalamus and the Control of Sleep

explain sleep-wake regulation

• sleep and wake are two stable states that switch in response to the build up of sleep pressure during wakefulness and the dissipation of sleep pressure during sleep

• sleep pressure accumulates during periods of wakefulness creating a drive to sleep, and then gradually dissipates during sleep allowing the transition back to wakefulness

• the switching state between sleep and awake is due to neurons that have antagonistic functions

what are the two types of hypothalamic neurons that regulate sleep-wake states?

• wake promoting neurons and sleep promoting neurons

what are wake-promoting neurons?

• neurons activated as sleep pressure dissipates, promoting the transition to wakefulness

what are sleep-promoting neurons?

• neurons activated by the accumulation of sleep pressure, promoting the transition to sleep

what is sleep pressure and the activity of the neuronal populations influenced by?

• various internal and external factors allowing the brain to adapt sleep-wake cycles to physiological and environmental needs

what are rapid bistable sleep-wake transitions controlled by?

• a distributed network of sleep and wake promoting neurons

where are the wake-promoting neurons found?

• in various brain regions, in the hypothalamus they are found in the lateral hypothalamus (LH) and the tuberomammillary nucleus (TMN)

what does the lateral hypothalamus (LH) contain?

• contains wake-promoting neurons that synthesise and secrete neurotransmitter hypocretin which drives and maintains the awake state

• when the neurons are activated, the LH makes neurotransmitter hypocretin promoting the awake state

what does the tuberomammillary nucleus (TMN) contain?

• contains another set of wake-promoting neurons that release neurotransmitter histamine, which also promotes wakefulness

what is the clinical importance of wake promoting neurons?

• lesion studies in rats have shown that damage to the lateral hypothalamus or the tuberomammillary nucleus disrupts wakefulness

• viral attacks to the LH or TMN, or a lack of their neurotransmitters (hypocretin from LH, and histamine from TMN) can cause narcolepsy

what is narcolepsy?

a disorder characterised by sudden uncontrollable falling asleep, an extreme case of being unable to stay awake appropriately

what was found in patients with narcolepsy?

• around 85-90% of patients with narcolepsy have a reduced number of hypocretin producing neurons in the lateral hypothalamus

• the loss of these hypocretin producing neurons in the lateral hypothalamus lead to excessive day time sleepiness and frequent day time napping, also can be associated with loss of muscle tone and paralysis

what was found when examining the control brain vs the narcoleptic brain?

• the control brain showed many cells in the latera hypothalamus producing hypocretin

• the narcoleptic brain showed little to no hypocretin producing cells in the lateral hypothalamus - showing a hypocretin deficiency in human narcolepsy

• comparison of normal versus narcoleptic brain shows a significant reduction in hypocretin neuron numbers, highlighting their critical role in maintaining wakefulness

what do Lhx9 expressing progenitors in the hypothalamus give rise to?

hypocretin producing neurons

what is Lhx9 TF?

• a master control regulator that binds to the gene encoding hypocretin

• it directs progenitors toward the hypocretin neuron fate

• it is required for hypothalamic progenitors to differentiate into hypocretin producing neurons

how was the developmental origin or hypocretin neurons traced in the study?

• a transgenic reporter line was created

• the enhancer/promoter region of the hypocretin gene was taken and placed upstream of a red fluorescent protein (RFP) gene

• in the transgenic animal, any cell that expresses hypocretin also expresses RFP

• this allowed cells to be visualised and tracked

how were hypocretin neurons isolated?

• the brains of zebrafish were dissected, dissociated into single cells and sorted using fluorescence activated cell sorting (FACS)

• this technique separated the RFP positive hypocretin neurons from all other non-fluorescent cells

what was done after isolating the hypocretin neurons?

• mRNA libraries were generated to identify transcripts expresses in the cells

• this analysis revealed several TFs upregulated in hypocretin neurons, including Lhx9

• Lhx9 was co-expressed and later confirmed as necessary for hypocretin neuron specification

why do the mRNA libraries need to be validated?

• to ensure that the genes of interest were actually expressed in the embryo

• aiming to confirm that Lhx9 TF is co-expressed in hypocretin positive neurons

what technique was used to validate the expression of Lhx9?

• in situ hybridisation performed on wild type embryos and on hypocretin EGFP transgenic line, examining the expression of Lhx9 and hypocretin

• cells expressing both Lxh9 (red) and hypocretin (green) appeared yellow, which indicates co-expression

• this validates that Lxh9 is present in the cells that produce hypocretin

how is temporal analysis of progenitor expression to determine whether Lhx9 is expressed in progenitors before hypocretin done?

• embryos can be analysed at different developmental stages

• early progenitor cells expressing Lhx9 but not hypocretin yet would later turn on hypocretin expression, confirming Lhx9’s role in specifying hypocretin neurons

how was it testes whether Lhx9 is sufficient to specify hypocretin neurons?

• a gain of function experiment was performed

• Lhx9 was cloned downstream of a heat shock activated promoter

• this transgene was then injected into zebrafish embryos, allowing overexpression of Lhx9 throughout the nervous system

• in these embryos, hypocretin neurons appeared not only in their normal locations but also in ectopic locations where hypocretin neurons do not normally develop

• this demonstrated that Lhx9 is sufficient to drive the specification of hypocretin positive neurons

how is the validation of ectopic hypocretin neurons done?

• the ectopic hypocretin positive neurons were examined with other markers that normally characterise hypocretin neurons, and they found that were found to have all the characteristics of hypocretin neurons

• the ectopic hypocretin positive neurons were also examined to see whether they could send their axons to their correct targets, and they did

• this shows that ectopic neurons generated by Lhx9 overexpression are indeed functional hypocretin neurons

how is necessity of Lhx9 for hypocretin neurons tested?

• through loss of function experiments

• knockout Lhx9 in embryos to visualise if hypocretin neurons would still develop

• two approaches can be used, splice blocking to reduce Lhx9 expression mRNA, or Cas9-mediated KO to remove Lhx9 gene

• as result of LOF experiments, hypocretin positive neurons failed to develop demonstrating that without Lhx9 these neurons are not generated

how does Lhx9 regulate hypocretin directly?

• overexpressing Lhx9 increases hypocretin

• mutating Lhx9 binding sites upstream of hypocretin gene prevents transcription

• this shows that Lhx9 directly activates hypocretin expression

what is a potential therapeutic approach for narcolepsy?

• cell replacement therapy

• using hypocretin expressing neurons in vitro is a potential therapeutic approach

• this approach makes it easier to understand the biology of hypocretin neurons and provides a platform for drug development, since human hypocretin neurons are difficult to isolate directly

• therefore creating these neurons in a dish is a crucial step towards targeted therapies for disorders like narcolepsy

what are induced pluripotent stem cells (iPSCs)?

• these cells are created by taking a differentiated cell and introducing specific TFs to reset the chromatin, reverting the cell to a stem cell like state capable of self-renewal and differentiation

• these iPSCs can then be cultured and exposed to different conditions to produce various specialised cell types, making them a powerful tool for research and potential therapies