L1 - SLEEP Flashcards
(38 cards)
1
Q
what is sleep
A
- a reversible behavioural state of perceptual disengagement from, and unresponsiveness to the environment
- reversible differs it from anaesthesia
- associated w/specific brain patterns/activities observed in EEG
- temporary loss of conciousness & decreased responsiveness to external stimuli; relaxed state
- muscle atonia in REM
- its an essential physiological process
1
Q
varying sleep times in mice
A
- different species sleep for different amounts of time
- engineered mice w/differing sleep time and structure but never one without sleep –> indicates that sleep serves a vital function
2
Q
Sleep in terrestrial animals
JM Siegel. 2005. Nature
A
- humans ~1/3 of lives asleep; other animals sleep even more, yet why we do remains a mystery
- sleep patterns vary widely; 18-20hrs in some species like bats/opposums; 3-4hrs giraffes/elephants
- sleep studies face challenges in quantification, often relying in visual observations and lacking systematic measurements
- correlations between sleep time, body mass & diet –> carnivores sleep most/ herbs least
- duration of sleep is correlated w/body mass and brain size, smaller animals have shorter sleep cycles –> not known why
3
Q
sleep in marine animals
JM Siegel. 2005. Nature
A
- unlike terrestrial animals, often exhibit unihemispheric sow waves during sleep
- one hemisphere = high-voltage EEG as it remains awake (eye open)
- no REM sleep –> maintain movement even during periods of SWA, challenging traditional definitions of sleep
4
Q
sleep stages (general)
A
- ~75% in NREM stages (N1, N2, N3); majority in N2 –> each stage = progressively deeper sleep
- 4-5 cycles per night; one complete cycle 90-110mins –> first REM period is short, as night progresses, longer REM and less time in NREM
5
Q
sleep stages- wake alert
A
- beta waves (12.5-30 Hz; highest F, lowest Amp) during eyes open phase –> desynchronised activity
- transition to alpha waves (8-12 Hz) when eyes close/ become more drowsy, slowly fade into N1
6
Q
sleep stages - N1
A
- 5% (1-5mins)
- light sleep
- theta waves (4-8 Hz)
- begins when more than 50% of a-waves become replaced with low amplitude, multiple frequency (LAMF) activity
- muscle tone present; regular breathing
7
Q
sleep stages - N2
A
- 45%
- heart rate & body temp drop
- sleep spindles = brief, powerful bursts of neuronal firing inducing Ca2+ influx into cortical pyramidal cells; mechanisms believed to be integral to synaptic plasticity & essential for memory consolidation
- K complexes = long delta waves (1s); maintain sleep & memory consolidation
- first cycle ~25 mins, lengthens w/each successive cycle
- where bruxism (teeth grinding) occurs
8
Q
sleep stages - N3
A
- deepest sleep (25%)
- delta waves (1-4Hz) = lowest F, highest Amp (slow-wave sleep, SWS)
- synchronised activity; neurons fire simultaneously
-most difficult to awaken from (>100 decibels); if awaken during N3, experience transient mental fogginess 0.5-1hrs after awakening - stage where body regenerates/ repairs tissues/ bones/ muscle, strengthens immune system
- when sleepwalking, terrors, bedwetting occurs
9
Q
sleep stages - REM
A
- 25%
- similar brain activity to wakefulness
- beta waves (12.5-30Hz) and desynchronized pattern
- associated w/dreaming
- not considered restful sleep
- muscle atonia (except eyes & diaphragm) & irregular breathing
- usually starts 90mins after sleep; each cycle increases (10mins - 1hr)
- tend to awaken spontaneously in the morning from REM
- increased O2 brain use; variable pulse & blood pressure; increased levels of Ach; brain is highly active (increase brain metabolism by 20%)
10
Q
what is an EEG
A
- non-invasive technique to measure brain waves/activity patterns associated w/different levels of conciousness
- records electrical activity in the brain
- place electrodes on scalp to detect & measure the electrical signals generated by neurons firing
11
Q
EEG invention
A
- Hans Berger
- cavalry man during WW1, fell off his horse
- before confirmation of his accident, sister dreamt about it
- Berger wanted to investigate telepathy: the possibility of transferring thoughts so aimed to use EEG to project thoughts & test if they could be detected by another person
- first person to observe & document regular electrical oscillations in human brain, which varied depending on levels of conciousness
- disproved telepathy
12
Q
Nathaniel Kleitman - “godfather of sleep”
A
- first human sleep labs in 1920s in U.chicago –> studying sleep & associated physiological processes in a controlled enviornment; analyse brain activity w/EEG during sleep
- found that sleep patterns can be affected by various factors: eg. mental illness, trauma –> changes in EEG in patients w/depression, schizo, PTSD
- was the groundwork for developing objective measures for evaluating sleep & sleep disorders
- prompted Aschoff & researchers in MUC sleep caves in 1960s/70s for circadian studies (total darkness/light)
- provided food at certain times of day to maintain some temporal organisation
- actogram over several days –> activity patterns follow a free-running rhythm w/each day’s activities starting slightly later than previous –> delayed onset of activity = endogenous circadian clock sits slightly longer than 24hrs
13
Q
how does the EEG work
A
- functionality = consistent across species primarily due to layered structure of the cerebal cortex (outermost layer responsible for higher functions)
- particularly important are pyramidal neurons
- neurons generate electrical signals when active –> fire action potentials
- active synapses allow the flow of ions (eg. K+) –> current flow between neurons
- the combined electrical activity of millions of neurons firing in synchrony creates detectable electrical signals picked up by electrodes in scalp (not individual neurons)
14
Q
EEG - wakeful state
A
- cortex is active –> processing sensory info, performing cognitive tasks, engaging in motor activities = irregular/ desynchronised activity
- reflects the dynamic and complex nature of cortical processing during wakefulness, w/ diff regions engaged in various tasks simultaneously
15
Q
EEG - transition to sleep
A
- NREM = more synchronised activity; slower oscillations of specific frequencies –> reflects the coordinated activity of large groups of neurons firing APs simultanously
- generating large EEG signals is most effective in structures w/many repeated neurons arranged in ordered layers (eg. hippocampus, neocortex)
16
Q
how to analyse sleep?
A
- FT to breakdown the complex brain wave signals into simpler F components
- allows to examine EEG in the F domain
- used to identify dominant brain rhythms; for functional connectivity analyses to explore communication between brain regions
17
Q
recording EEG in mice
A
- neurologger = lightweight helmet –> can be surgically implanted onto mouse’s skull (chronic implantation allows for long-term recordings)
- key advantage = assess sleep in patterns over extended time periods in natural environment; insights into interactions w/ other physiological processes
- mice = model organisms because many of the fundamental processes that regulate sleep are conserved across mammalian species (incl. basic architecture of sleep stages; neural circuits involved in sleep-wake regulation; molecular mechanisms underlying S and C)
- can create animal models of sleep disorders via manipulating genes/circuits –> allows researchers to characterise the alterations in sleep patterns & brain activity associated w/these disorders & develop potential treatments / interventions
18
Q
sleep in mice
A
- polyphasic sleep patterns –> sleep in multiple bouts throughout the 24hr-period
- nocturnal animals; active in dark phase; naps in light phase
- most sleep is NREM
19
Q
local field potential
A
- placing electrodes into cortex –> samples activity of 1000s of neurons in its vicinity
-analogous to EEG - NREM = regular activity ~4Hz oscillations = synchronised neural firing patterns in the cortex
20
Q
multi-unit activity (MUA)
A
- recording firing patterns of individual neurons during sleep in Rasta plots (red bars = APs)
- depolarization - increase likelihood of firing AP
- during wakefulness, neuromodulators such as histamines/acetylcholine maintain cortical activity; in NREM = these are reduced
21
Q
physiological changes during sleep
A
1) cardiovascular = increase heart rate/ blood pressure upon wakening; increases risk of myocardial infarction in the morning
2) sympathetic nerve activity decreases during NREM, increases in REM
3) faster and more irregular breathing during REM; more regular in NREM
4) NREM = lower cerebral blood flow & metabolism, increase in REM
5) renal = decrease excretion of electrolytes = concentrated urine
6) endocrine = growth hormone secretion peaks during early sleep stages; melatonin induces sleepiness
22
Q
recovery sleep after sleep deprivation
A
- longer & deeper NREM sleep after deprivation –> body has compensatory mechanisms to recover from sleep debt + restore homeostasis
- not known why
23
Q
novel object experiment
A
1) baseline monitoring = sleep patterns & behaviour; EEG; EMG; video –> reference point for comparing changes during sleep deprivation
2) sleep deprivation is induced by introducing novel objects into the environment at regular intervals (eg. every 1hr for 6hrs) –> timing and frequency varies depending on experiment; have to ensure animal remains engaged
3) continously monitor behaviour/ sleep/ wakefulness through EEG, EMG, video
4) sleep recovery phase = allowed to rest undisturbed in cages –> continous assessment
24
ecological reasons to sleep/ not sleep
1) predation risk = eg. wild cats sleep more to conserve energy for hunting; prey animals sleep less as they have to remain vigilant & detect / evade predators
2) foraging opportunities & food availability
3) environmental conditions (eg. temp, humidity, sunlight) = adjust sleep patterns seasonally or during migration
4) sleep flexibility = eg. some marine mammals have unique adaptations allows them to sleep w/one hemisphere whilst the other remains awake
25
tools used in neuroscience research - in situ hybridisation (ISH)
- technique to visualise & localise specific nucleic acid sequences within tissues / cells --> eg. can study neurons in the hypothalamus to elucidate molecular mechanisms underlying sleep regulation
- use nucleic acid probes complementary to specific mRNA transcripts of interest --> designed to target genes known to be involved in sleep regulation (eg. neurotransmitters, receptors, enzymes, etc)
- probes are labelled w/ detectable markers (eg. fluorophores/ radioisotopes) to visualise mRNA expression patterns within the tissue
- ISH on hypothalamic tissue; prepare brain slices; treatment to permeabilise cell membranes & denature nucleic acids, allowing probes to hybridise specifically to their complementary mRNA; excess probes are washed away & tissue sections are examined under the microscope; can identify & characterize expression patterns of genes in different neurons
- combined w/ other techniques eg. immunohistochemistry, scRNA-seq; for insight into molecular & cellular diversity of neurons involved in sleep regulation
- correlate mRNA expression patterns w/other phenotypic characteristics (eg. neurotransmitter content or connectivity patterns) --> deeper understanding of how different neuronal populations contribute to the complex regulation of sleep-wake behaviour
26
tools used in neuroscience research - activity tagging
c-fos promoter:
- regulatory region of c-fos gene; becomes active in response to elevated levels of intracellular Ca2+
- when neuronal activity increases, Ca2+ levels increase so TFs bind to c-fos promoter initiating transcription of c-fos gene --> fos protein which serves as a TF itself, regualting the expression of other genes
chemo-genetic receptors:
- eg. hM3Dq; synthetic receptors enginereed to respond to artificial ligands (eg. CNO)
- typically fused to reporter proteins (eg. mCherry) allowing for visualisation & tracking of cells expressing the receptor
AAV delivery:
- c-fos promoter driving the chemogenetic construct is packaged into AAV vector which is inserted into brain region of interest
- they efficiently transduce a wide range of cell types which is why its widely used
- small, non-enveloped Parvoviridae virus; harmless; safe + efficacy; requires helper virus (eg. HSV) to replicate & produce viral particles
- elicits minimal immune response (particularly T cells) --> reduces risk of immunogenecity & allows for repeated administration if necessary
- key advantages = ability to mediate long-term gene expression in target cells; once delivered into host cell, therapeutic gene carried by AAV can integrate into the host genome or persist episomally, allowing for sustained expression of the transgene over an extended time period
Tet-activator system for temporal control:
- c-fos promoter is constitutively active to an extent due to basal levels of Ca2+ in neurons, so have to regulate the expression of chemo-genetic receptors until the experiment
- tet activator = system from E. coli = binary control mechanism for gene expression
- administer doxy (tetracycline derivative) to repress the tet activator, preventing the transcription of chemo-genetic receptor gene until doxy is removed
27
pharmacogenetics
- by introducing genetic modifications you can design receptors that selectively interact w/synthetic compounds or drugs of interest
- engineered receptors are then expressed in target neurons, allowing for precise pharmacological modulation of their activity w/o affecting other neuronal populations
28
pharmacogenetics - CNO
- clozapine-N-oxide = compound used in neuro research as an inert control for clozapine (drug used in psychiatry)
- can activate DREADDs when they are genetically expressed in neurons
- receptors used in DREADD are mutant muscarinic receptors engineered to not respond to their endogenous ligand (Ach), but instead are activated by CNO
29
pharmacogenetics - engineering DREADD receptors
- hM3Dq (excitatory) & hM3Di (inhibitory) --> GPCRs
- using yeast-based natural selection techniques, researchers modified the aa sequence of the receptors to confer sensitivity to CNO and abolish responsiveness to Ach
- often fused to fluorescent proteins to visualise expression of DREADD receptors
30
pharmacogenetics - DREADD receptors pharmacological modulation
- upon activation by CNO, hM3Dq signals through Gq pathway --> activation of intracellular cascades such as inositol metabolism & PKC activation resulting in neuronal excitation
- hM3Di --> opening of K+ channels & inhibition of cAMP signalling = neuronal inhibition
- pharmacological manipulations allow to selectively activate/inhibit specific neuronal populations in vivo, facilitating the study of neuronal circuits & behaviour
31
what controls sleep?
- two process model of sleep is the widely accepted framework --> describes the interaction between S & C, proposed by Alexander Borbeley in the 1980s
32
what controls sleep? - homeostatic regulation
- the drive for sleep; accumulates as we wake
- body's need for sleep based on prior wakefulness & deprivation
- as an individual remains awake, adenosine neurotransmitter accumulates in the brain, resulting in increased sleep pressure
33
what controls sleep? - circadian regulation
- animals have specific sleep patterns influenced by environmental cues (eg. light) + internal biological rhythms --> synchronise these to help animals anticipate events like feeding & metabolism
- C is driven by internal biological clock in the suprachiasmatic nucleus (SCN) of the hyppthalamus --> regualtes the timing of various physiological & behavioural processes over a 24hr period
- SCN receives input from light-sensitive retinal ganglion cells, synchronising the body's internal clock w/ external light/dark cycle --> promote wakefulness during day & sleep at night
- circadian rhythms influence body temp, hormone secretion, etc
34
what controls sleep? - interaction between S & C
- S regulates sleep intensity & duration
- C regulates timing of sleep & wakefulness
- propensity of sleep is highest when both processes align eg. at night, when its dark & sleep debt accumulates
- disruption to either process (eg. irregular sleep schedule, jet-lag, shift work) can lead to sleep disturbances & disorders
- neurotransmitters (eg. serotonin, norepinephrine, histamine, orexin) are involved in maintaining wakefulness & modulating transitions between sleep stages
- SCN regulates rhythmic expression of clock genes & secretion of melatonin by the pineal gland, helps signal onset of darkness & promote sleep
35
what controls sleep? - motivational regulation
- Rothschild et al. 2017. Curr Opin. Neurobiol
- foraging, mating, predator presence, all influence arousal levels & sleep behaviour in the wild
eg. polygynous pectoral sandpipes (males) reduce sleep to as little as 2hrs/day during mating periods --> had highest breeding success
eg. great frigatebirds sleep only 0.7hrs/day during foraging flights, whilst 12/8hrs on land
- arousal response to environment & homeostatic factors involve neruonal mechanisms centered around the LH orexin neurons --> implicated in increasing wakefulness during stressful conditions e.g reduced food availability
- orexin neurons produce 2 neuropeptides (hcrt1/2) that project to various sleep/wake reguatory nuclei expressing hcrt receptors
- transgenic mice lacking hcrt fail to increase wakefulness/locomotion following fasting
- hcrt neurons play role in regulating reward by modulating VTA dopaminergic neurons --> key regulators of motivation & sleep/wake states
- inhibition of VTA dopaminergic neurons prevents maintenance of wakefulness even in predator scent, potential mates, etc
- animals engage in specific behaviours to prepare for sleep: shelter from predators, postures, etc --> need to suppress neurotransmitter systems/ brain circuits associated w/ wake-related behaviours
36
sleep as a problem of localisation
- stems from observations made during the spanish flu pandemic WWI, which had significant neurological rammifications for affected individuals
- von Economo's research on brains of patients w/ encephalitis lethargica, disorder characterised by profound sleep alterations
- identified specific brain regions associated w/sleep regulation --> lesions in hypothalamus & brainstem were often correlated w/ changes in sleep patterns & arousal states; suggests that sleep could be localised to distinct anatomical structures within the brain
- functional MRI (fMRI) & PET scans used to visualise brain activity during different stages of sleep & wakefulness w/unprecedented precision --> found distinct patterns of neuronal activity in various brain regions during REMS & NREMS (eg. heightened activity in hypothalamus & brainstem during REMS; decreased activity in PFC during REMS)
- studies employing animal models (eg. rodents) --> insights into neuronal circuits & neurotransmitter systems governing sleep regulation
- chemogenetics & optogenetics facilitated the selective activation/ inhibition of specific neuronal populations, elucidating their contributions to sleep-wake behaviour
- neurons in VLPO release inhibitory neurotransmitter (GABA & galanin), which suppress arousal-promoting regions in brainstem & hypothalamus facilitating transition from wakefulness to sleep
37
the hypothalamus
- located at the base of the brain --> crucial role in regulating a wide range of physiological processes
- contains specialised nuclei (VLPO, SCN) involved in the regulation of sleep-wake cycles
- helps maintain body temperature --> regulates sweating, shivering, vasodilation/ constriction
- certain nuclei (arcuate nucleus in LHA) play key roles in regulating appetite, hunger, and thirst --> nuclei integrate signals from the body's metabolic state & regulate feeding behaviour accordingly
- produces & releases various hormones
- while the size & complexity of the cortex has expanded significantly in humans compared to other mammals, the hypothalamus remains relatively conserved in size and structure across species
