Physiology of Sleep

Question 1

1- definition of sleep
2- what does sleep differ from, how?
3- what is it?

Answer 1

1- Sleep is a natural, periodic state (happens at a regular time period) that involves reduced responsiveness to environmental stimuli (when were asleep we don’t pay attention to whats going on around us) and decreased mobility

2- Sleep differs from states such as coma, vegetative state or brain death
— It is reversible
— Self-regulated

3- It is a behaviour observed in humans across cultures and across species

Question 2

How is sleep studied in humans vs animals

Answer 2

In humans: Subjective and Objective Measures

In animals: Objective measures

Question 3

Studies in Humans:
subjective vs objective measures

Answer 3

Subjective Measures:
- clinical interviews- ask people questions
- sleep diaries and dream diaries- gold standard way of getting subjective information about peoples sleep/ dreams
- questionnaires and scales

Objective Measures:
- actigraphy- using phones/ watches
- polysomnography (PSG)- recordings of EEGs that have been used to record brain activity during sleep
- PET
- fMRI/ EEG

Question 4

Polysomnography
1- what is it?
2- discovered by? when?
3- where is it used?
4- what does it involve?
5- what type of tool

Answer 4

1- The ‘gold standard’ of sleep research
2- Discovered by Hans Berger 1929
3- Used in several species for research and clinical purposes
4- Involves the placement of electrodes on the scalp and face to allow recordings of electrical activity from multiple areas, mainly cortical
5- non evasive tool

Question 5

List the type of polysomnography recordings

Answer 5

Recordings from multiple sources

EEG recordings (electroencephalogram): recordings from populations of neurons in the brain underneath the skull (get recordings from multiple neurons instead of a single one)

EOG recordings (electrooculogram): recordings from the muscles around the eyes

EMG recordings (electromyogram): recordings from the muscles

• These recordings can be combined with measures of heart rate, temperature, breathing (O2) etc.

Question 6

Polysomnography:
1- disadvantages
2- what effect can be evident?
3- what do EEGs need to be grouped into?
4- recording time?

Answer 6

1- It is costly and requires a special place (sleep lab) and trained personnel
- Although not invasive there may be some discomfort involved
2- First-night effect (for a lot of people changing places/ bedrooms/ on holiday means were not doing our normal regular sleep therefore its usually disrupted on this first night)
3- Need to group EEGs into frequency bands
4- Requires scoring in short periods of time called epochs (i.e. 30-second epochs)

Question 7

Sleep recordings:
1- what does careful analysis of brain activity and other features reveal?
2- what types of sleep are most commonly talked about?
3- stages?

Answer 7

1- sleep architecture
2- NREM and REM
3- Three stages of sleep (NREM) and an additional REM sleep episode: Stage 1 (N1), Stage 2 (N2), Stage 3 (N3) and REM sleep
*initially 4 distinct stages of NREM and one REM but then revised to 3 by the American Academy of Sleep Medicine (AASM)

Question 8

Brain activity during wakefulness:
how can neuronal activity observed in the EEG during wakefulness vary?

Answer 8

Beta waves consist of irregular activity of 13–30 Hz (and sometimes gamma waves 30+ Hz- even faster)
— Beta activity takes place when the brain is processing information
— The person is alert and attentive to events in the environment or engaging in cognitive processes

Alpha waves consist of activity of 8–12 Hz.
- Occur when a person is resting quietly, not particularly aroused or excited and not engaged in strenuous mental activity

Question 9

Brain activity during sleep
First 2 steps

Answer 9

• Sleep begins with a state of relaxation, feeling drowsy
• Stage 1 (N1) (3.5–7.5 Hz): presence of theta activity - a transition between sleep and wakefulness. Usually lasts 1-2 min
• A unique feature is the slow eye movements (SEMs) where the eyes roll back and forth slowly (2-4s to move back and forth) and their occurrence seems to coincide with our drifting of awareness (Porte, 2004)

Question 10

Brain activity during sleep
Next step

Answer 10

Stage 2 (N2) : Sleep begins – irregular activity with sleep spindles (12-14Hz) although these occur in other stages of sleep and K complexes which are only found during stage 2. We spend more time in this stage at night.
(sleep spindles- to do with things such as synaptic plasticity and learning, K complexes- to do with things such as disengaging us from environmental stimuli going on around us)

Question 11

Brain activity during sleep
Final step

Answer 11

Stage 3 (N3): High-amplitude and low-frequency delta activity (less than 3.5 Hz) or even slower of less than 1Hz (slow oscillations)
- Synchronized, regular waves, reflecting synchrony and coordination of activity of neurons in underlying brain areas
- Brain activity and other bodily functions (heart rate, breathing, kidney function, etc) slow down.
- These stages are collectively referred to as slow-wave sleep [deep sleep]

slow firing of the neurons but they are synchronising very slow activity (like an orchestra)

Question 12

REM Sleep

Answer 12

• A sleep phase characterized by increased brain activity, eye movements (left and right) and asynchrony in brain waves accompanied by muscle atonia
• Aserinsky and Kleitman,1953: Sleep characterized by rapid eye movements - Rapid Eye Movement sleep (REM)
• Michel Jouvet, 1959: Deep sleep, in terms of muscle activity but light sleep, in terms of increased brain activity, muscles are paralysed - Paradoxical sleep
• Twitches, penile erections, vaginal secretions and dreams also occur during this stage

Question 13

Discovery of REM sleep
3 time frames

Answer 13

• Eugene Aserinksi (1951) a graduate student recorded his 8y old son’s eyes using a dynograph to detect his son’s blinking and know that he was awake
• In 1953 he published with his advisor, Nathaniel Kleitman in the journal Science the paper “Regularly Occurring Periods of Eye Motility and Concomitant Phenomena, During Sleep” (Reprinted in The Journal of Neuropsychiatry, 2003)
• 1957 – Dement & Kleitman discovered the connection between these eye movements and dreaming
  – Recorded from 9 adults who reported 351 dreams
  – Dreaming during REM 80% vs 7% during NREM- this seemed like a big difference and they had the conclusion that the phase where our eyes are moving a lot and other things are happening is when they are dreaming
  – Thus, dreaming became a biological phenomenon
  – they had polysomnography recording on one hand

Question 14

M. Jouvet (1965)

Answer 14

• At a symposium in 1965, many were emphasizing the similarities between NREM and REM sleep and sleep as a unitary phenomenon
• Dement commented that synchronized and REM sleep..

“are as far as night and day. It is difficult to point to a single attribute that is commonly shared…in terms of definition it would seem more appropriate to regard slow-wave sleep and paradoxical sleep as entirely different states with their own specific mechanism or mechanisms. I would even go so far as to suggest that there may be some validity in questioning whether they should be subsumed under the general heading of sleep”
(from Steriade and McCarley, 2005, p.9)

Question 15

Study of sleep in animals:
1- primarily based on recordings of electrical activity in animals that are …
2- allow for? + 2 examples
3- what manipulations?

Answer 15

1- Primarily based on recordings of electrical activity in animals that are awake or asleep
2- Allow for greater manipulations: pharmacological, anatomical and behavioural etc
– Allowed for lesions- destroying a particular part of the brain to identify its function, transections (cuts) and pharmacological interventions
– Brain tissue evaluation (staining or labelling specific proteins, etc)
3- Genetic manipulations- you can silence or activate genes and then see if that particular gene has any affect on the behaviour you are observing

Question 16

Studying Sleep in Animals:
1- who are used?
2- what are rats vs humans?
3- contributions of animal research?

Answer 16

1- Initially cats, but now we primarily use rats and mice, flies are increasingly used as well
2- Rats and mice are nocturnal (active during dark hours and asleep when lights are on) vs humans are diurnal
2- Rats are polyphasic (only have a couple of hours of sleep) when it comes to sleep whereas humans are monophasic
3- Animal research has contributed massively to the advancement of the field

Question 17

Traditional vs Telemetry Recordings

Answer 17

Example of recording when animals were pressing a lever to get different stimulations

• they would have electrodes that were planted in their skull
• nowadays we can implant a transmitter under their skin so it is a simpler procedure
• transmitter sends information about activity to receiver
• can track movement/ temperature
• can record electrical activity when they are asleep as well as when they are awake

Question 18

Recordings from a rat: waking, SWS, REM

Answer 18

Waking:
- The EEG is the activity of the brain when the rat was awake- shows there is brain activity
- The EMG is the muscle activity- shows there is some muscle activity but it is quite low

SWS
- everything is slowing down (temperature drops, breathing drops, heart rate slows down)
- muscles are very relaxed (EMG)

REM:
- electrical activity that is similar to wakefulness but there is no muscle activity

Question 19

Gradual emergence of REM features in a rat

Answer 19

Birth
P0 Limb twitches (thought o aid motor development)
P1 Muscle atonia
P4 Hippocampal theta
P6 Rapid eye movement
P10 Cortical activation
Eye opening

(Things are not all happening at once, they are happening gradually)

Question 20

Challenges to the standard definition of REM
Using animal examples

Answer 20

Platypus:
in line- rapid eye movements, bill twitching
not in line- but no EEG activation, instead, shows cortical slow waves typical of non-REM sleep

Goose:
in line- rapid eye movements, twitching EEG activation
not in line- but not muscle atonia when the head is not supported

Owl:
in line- twitching, EEG activation, suspended thermoregulation
not in line- but not rem or muscle atonia

Armadillo:
in line- rem, twitching, EEG activation, hippocampal theta, muscle atonia
not in line- but penile erections occur during non-REM sleep

Tegu:
in line- rem
not in line- but not twitching or wake-like brain activation, also as an ectotherm, cannot suspend thermoregulation

Question 21

REM Microstates in Humans
1- what are microstates typically called
2- what does fMRI distinguish between
3- what phase is response to external stimuli greater in?
4- what is enhanced during phasic REM?

Answer 21

1- Microstates (microstructures) typically called tonic REM and phasic REM states
2- fMRI distinguishes well between these two phases and suggests that REM can be ‘shallow’ or ’deep’
3- Response to external stimuli is greater during the tonic phase of REM
4- During phasic REM there is enhanced thalamocortical synchronized activity

Question 22

Phasic and Tonic REM microstates:
How do these differ?

Answer 22

The difference is to do with the eye movements:
- Phasic: a lot of eye movements, irregular heart rate.
- Tonic: still rem but eye movements are more quiet and there is a bit more regular heart rate

Question 23

Wehrle et al 2007
Brain reactivity to acoustic stimulation during the two phases of REM and thalamocortical activity

Answer 23

They put acoustic stimulation in MRI. They were giving them signals in their ears

They were more responsive in tonic phase but less responsive/ not responsive at all when they were in the phasic.

Paradoxical Phasic REM:
Greater cortical activation and autonomic arousal, but also decreased responsiveness to external stimuli compared to tonic REM

To sum: In tonic phase, brains responsive to acoustic stimulation

Question 24

Is sleep an active or passive state? (this is an unanswered question)

Answer 24

• Lucretious, 1st ce BC – Considered sleep a passive state and wake an active process
• Idea that sleep occurs passively with decreases in sensory input (arousal diminishes)

Question 25

Early Observations of sleep
Name of neurologist and what he reported

Answer 25

Viennese neurologist Baron Constantin von Economo evaluated patients with encephalitis lethargica- they called it this because many patients were sleeping a lot and waking up just to eat and sleep. Whatever was going on in the brain was affecting their alertness.

In 1918 he reported:
— Most patients had continuous sleepiness- slept more than 20 hours per day and would wake up only to eat and drink

Fewer patients displayed insomnia (couldn’t go to sleep)

Question 26

What did Frédéric Bremer (Bremer, 1938) do?

Answer 26

A- CERVAU ISOLÉ
cut a bit towards the front and the animals displayed SWS only (forebrain)

B- ENCEPHALE ISOLÉ
cut more towards the body animals would display all states (brain)

Question 27

What did von Economo (1930) do?

Answer 27

With patients with encephalitis lethargica- when they would die Economo would look at their brains and see that patients had a lot of damage in their brains and cells that were dying due to the infection. They were displaying hyposomnolence higher up vs if the area infected was more towards the basal forebrain, in this case they were displaying insomnia.

Insomnia higher up than Hypersomnolence

Question 28

Cutting the brain of cats results and main takeaway (Bremer, von Economo)

Answer 28

Cut the brain of cats at different levels:
— When transection was made between the medulla and the spinal cord (isolated brain), the animals displayed wake, SWS and REM sleep. Thus, these functions were seated in the brain and not the body
— When the transection was made in the midbrain (isolated forebrain) the animals displayed constant SWS. Thus, SWS is promoted by the forebrain but arousal and REM require other areas in the brainstem.

Conclusion: Brain and wakefulness are controlled somewhere in the brain but there are probably different areas that are controlling wakefulness and sleep because different cuts are explaining different types of behaviour.

Question 29

The sleep centre:
What did Nauta (1946) do?
What did he conclude?

Answer 29

• Conducted a series of lesions in rats and concluded: “There is a reason to accept a structure in the preoptic region, which is of specific importance for the capacity of sleeping (“sleep centre”)” (Nauta, 1946)
• Most likely sleep is caused by an inhibitory action of the sleep center on the waking centers - two centers are trying to antagonise/ silence each other and who wins will determine which state they will be in
• He concluded that this area is around the basal forebrain and specifically around the preoptic area of the hypothalamus
• McGinty and Sterman (1968) verified Nauta’s finding in cats.

Question 30

What did Szymusiak and McGinty (1986) do and find?

Answer 30

Recorded from neurons in the basal forebrain during waking, NREM and REM states

A few neurons were firing during wakefulness but most neurons were firing during non rem sleep

Question 31

What did Sherin et al 1996 do and find?

Answer 31

• Used immunocytochemistry to identify FOS protein in a group of vlPOA neurons.

— immunocytochemistry is a specific way to target proteins- knowing the protein structure you can create antibodies which find this protein (eg. receptor/ neuron) and it attaches to it and a second antibody can be tagged and given a colour to actually see the neuron has the receptor/ protein were looking for and visualise it under the microscope later on - in short- a way to label proteins and look at them under the microscope and we can quantify/ measure how mnay neurons have this tag so we can tell if they have this particular tag so we can tell that they have this particular protein.

— in this case fos is a protein that indicates that the neuron before the animal was sacrificed was active (firing APs)

• Rats sacrificed during their dark phase (little sleep) had about 4 active cells vs rats sacrificed in the light phase (a lot of sleep) had about 28 active cells

Question 32

FOS immuno-reactivity in the vlPOA Sherin et al (1996)

Answer 32

Graphs of FOS-immunostained coronal sections through the preoptic hypothalamus of spontaneously behaving rats that slept 15%, 63%, a sleep deprived rate that slept 83% of the hour before they were killed

FOS-ir cells are apparent in the VLPO of animals that slept most of the hour before they were killed.

For 83% rats- dark sports were previously active- these were asleep most of the time before they were sacrificed

So fos was able to narrow things down to this region as being the region which is involved in NREM sleep. These cells are primarily gabaergic.

Question 33

What is the sleep centre?

Answer 33

VlPOA

Question 34

vlPOA and mnPNA:
1- what do they both contain?
2- what is mnPN more responsive for?
3- what is the vlPOA cluster necessary for?
4- what does damage to VLPOA cause?
5- what does electrical stimulation of vlPOA cause?

Answer 34

1- The vlPOA and the mnPNA both contain ‘sleep-active’ neurons (when were asleep these neurons are active/ firing)
2- The mnPN is more responsive to the homeostatic drive for sleep
3- The vlPOA cluster is necessary for sleep, especially NREM sleep (Lu et al, 2000 JNeurosci)
4- Damage to this area causes insomnia in rats and they eventually die (survive for only three days)
5- Electrical stimulation of this area causes drowsiness and sleep

Question 35

What has proven to be a sleep promoting factor

Answer 35

Cerebral spinal fluid

Question 36

Adenosine:
1- morning vs evening?
2- what release adenosine and impact of this?
3- what does adenosine do?
4- what do adenosine agonists do?
5- what do adenosine receptor antagonists do?

Answer 36

1- Accumulates during the day (little in the morning as our neurons are well rested), after prolonged wakefulness and increased need for glycogen (energy stores)
2- Astrocytes release adenosine to inhibit neuronal activity (to tell our neurons to slow down)
3- Adenosine promotes sleep, which allows for the restocking of glycogen
4- Adenosine agonists decrease wakefulness and increase sleep, favouring SWS
5- Adenosine receptor antagonists increase wakefulness and decrease sleep

Question 37

Adenosine and Homeostatic Regulation of Sleep (Benington et al 1995)
awake / SWS / caffeine

Answer 37

when awake: astrocyte is releasing adenosine and binding to the adenosine neuron to slow it down

SWS: this is cleared, when we go to sleep adenosine is cleared/ taken up. But when we are awake for a long time adenosine increases

caffeine: blocks adenosine receptors. Caffeine acts on A1 and A2A receptors to antagonise the effects of adenosine.
Coffee will sit on the receptors for adenosine- adenosine is there but it cannot act and make you sleepy. Although we can become tolerant.

Question 38

What promotes sleep? (+ explain)

Answer 38

Melatonin

• the ‘dracula’ of hormones as it is secreted in the evening
• produced and secreted by the pineal gland in the centre of the brain

Question 39

Wakefulness and Arousal
1- who discovered these regions accidentally
2- what did they notice when recording anesthetized cats?
3- what did stimulating the brainstem in a sleeping cat cause?
4- evidence for an active waking centre coined the …..
5- what has ARAS been considered essential for?

Answer 39

1- Moruzzi and Mogoun (1949)
2- they stimulated the cat’s brainstem they noticed that the delta waves were replaced by beta waves
3- resulted in waking the animal
4- “ascending reticular activating system” (ARAS)- small structures in the back of the brain that are contributing to wakefulness- projecting from base of the brain higher up to make us more alert and wakeful
- arousal and wakefulness since

Question 40

The Ascending Reticular Activating System (ARAS) (Aston-Jones and Bloom, 1981)
1- what is it
2- what do these nuclei do?
3- what does the dorsal branch consist of?
4- when are these active?

Answer 40

1- a network of nuclei that use different neurotransmitters
2- These nuclei project to the cortex via a dorsal and ventral branch to promote arousal

Question 41

ARAS dorsal vs ventral branch

Answer 41

The dorsal branch consists of the Pedunculopontine (PPT) and Laterodorsal tegmental nuclei (LDT) that use primarily Acetylcholine (Ach)
*These are active during both wakefulness and during REM

The ventral branch includes the:
- Locus Coeruleus (LC; norepinephrine or noradrenaline NE/NA)
- Ventro-lateral periaqueductal gray (vlPAG; dopamine, DA)
- Raphe nucleus (RN; serotonin, 5HT)
- Tuberomummillary nucleus (TMN; Histamine)
*All of these fire more during wakefulness than during NREM sleep and show no activity during REM sleep

Question 42

Brain regions promoting wakefulness

Answer 42

Lateral hypothalamus (ORX)
Basal forebrain with acetylcholine

In contrast to this, vlPOA sends projections to all structures and inhibits their activity

Question 43

Name a neurotransmitter that promotes alertness and wakefulness

Answer 43

Acetylcholine

Question 44

Acetylcholine (ACh):
- where is it produced
- what do ACh agonists increase?
- what did Marrosu et al 1995 discover?

Answer 44

Produced mainly in two areas:
- Nucleus basalis of Maynert (NBM) in the basal forebrain
- In the pons, the pedunculopontine tegmental nucleus (PPT) and the laterodorsal tegmental nucleus (LDT)

• ACh agonists increase EEG signs of cortical activity
• Marrosu et al 1995 – microdialysis (involves getting some csf and you analyse it to see how mnay neurotransmitters are available/ ….. watch) to measure ACh in the hippocampus and cortex and found that ACh was high during wake and REM sleep (desynchronized activity) and low during SWS

Question 45

Release of Acetylcholine during the Sleep/ Waking Cycle
(Marrosu et al., 1995.)
Cortex vs Hippocampus

Answer 45

Cortex graph:
Acetylcholine is higher when awake and during REM but there is not much of it in SWS

Hippocampus graph:
Acetylcholine is medium during waking and then low during SWS and high in REM

Question 46

Norepinephrine (Noradrenaline)
1- where is it produced?
2- what does increase and correlates with?
3- axons from the LC project where?
4- What did Aston-Jones and Bloom (1981) do?

Answer 46

1- Norepinephrine is produced in the locus coeruleus (LC) located in the dorsal pons
2- Norepinephrine increases vigilance and correlates with performance in a cognitive tasks
3- Axons from LC project throughout the cortex, hip, thalamus, cerebellum, pons and medulla
4- Aston-Jones and Bloom (1981) – recorded from LC neurons across sleep and waking of rats
- during wakefulness neuronal activity was high, during SWS activity was low and during REM it was close to zero

Question 47

Norepinephrine during wakefulness and sleep

Answer 47

During wakefulness there are a lot of norepinephrine firing but then they decrease more in SWS and there is not much activity in REM but then they start to increase again when waking.

Question 48

Serotonin (5HT)
1- where are Serotonergic neurons found?
2- facilitates?
3- when is activity of serotonergic neurons high?

Answer 48

1- Serotonergic neurons are found in the Raphe nucleus in the pons and medulla and project to many regions in the brain
2- Facilitates ongoing (perhaps repetitive movements, such as grooming, chewing etc) and not novel stimuli
3- Activity of serotonergic neurons is high during waking and declines during SWS and reaches almost zero during REM

Question 49

Serotonin during wakefulness and sleep

Answer 49

Serotonin is active when awake but then it gets reduced when in SWS and REM but as soon as waking again it goes up

Question 50

Histamine:
1- where is it synthesised?
2- increases?
3- high vs low activity?
4- what does suppression/ decrease of histamine cause?

Answer 50

1- Synthesized in the tuberomammillary nucleus (TMN)
2- Increases cortical activity and arousal and also increases ACh release in the cx
3- High activity of histamine neurons during wakefulness but low during SWS and REM sleep
4- Suppression of histamine or blockade of its receptors decreases wakefulness and increases sleep (antihistamines- they battle histamine and therefore make you sleepy)

Question 51

The flip-flop switch Cliff Saper 2001

Answer 51

• either on or off
• describes the antagonistic nature of sleep and wake centres
• they are mutually inhibitory- they both try to silence each other
• “on”: arousal centre is activated because the vlPOA is being inhibited
• “off”: vlPOA is inhibiting the arousal centres and induce sleep

Question 52

Orexin or Hypocretin
1- what is it and released from where?
2- responsible for?
3- implicated in?

Answer 52

1- It is a peptide released from the lateral hypothalamus (LH)
2- Highly responsible for the maintenance of wakefulness
3- Implicated in narcolepsy

arousal transmitter which helps to stabilise the flip-flop switch- it doesn’t happen in people with narcolepsy as the other pressure of orexin is not there

orexin is important in maintaining arousal

Question 53

Orexin (or Hypocretin)
1- what, produced where?
2- involved in?
3- how many neurons in the human LH
4- what did Mileykovskiy et al 2005 do?

Answer 53

1- Peptide produced in the lateral hypothalamus (discovered in 1998)
2- Involved in narcolepsy – degeneration of orexinergic neurons in humans or hereditary absence of one type of receptor in dogs
3- About 7000 neurons in the human LH - their axons project to almost every part of the brain including the cerebral cortex, LC, raphe, TMN and ACh regions and exert an excitatory effect (arousal)
4- Mileykovskiy et al 2005: did single cell recordings from orexin neurons in rats and found that they fired at a high rate during alert and active waking and at a low rate during quiet waking, SWS and REM. Highest rate of firing during exploratory activity

Question 54

Orexin during sleep and wakefulness

Answer 54

High during wakefulness then silent in SWS and REM and begins to increase again for waking. There is no imbetween here.

Question 55

Another depiction of the flip-flop switch

Answer 55

Think about it as a seasore

If we are awake- all the NTs in the arousal system are active and inhibiting the vlPOA. The LH with orexin is also stimulating these areas so we cannot sleep at this time.

Then vlPOA is gaining ground and inhibiting the LH, TMN, DR, LC

Question 56

Flip-flop switch in rem

Answer 56

When we are asleep we are inhibiting the rem off so we may easily go into rem sleep.

When rem on is activated it will inhibit movement and eye movements may happen.

If we get startled/ frightful this may lead to rem on to go on and then a person with narcolepsy might go into rem.

(this content above we are not expected to know as well)

Question 57

A typical night

Answer 57

• go through stages (1, 2, 3)
• 3 is SWS and deep sleep
• then we start to come up
• have REM sleep
• go into deeper sleep
• have another REM
• most of our sleep we spend in stage 2 but this varies depending on the time of the night
• when we first go to sleep- spend more time in stage 3 (SWS) but then you might not have another SWS episode later in the night. Opposite happens with REM- short episodes at the start of the night but longer when the night goes on.

Proportion of total sleep time per stage:
N1= 2-5%
N2= 45-55%
N3= 20%
REM= 20-25%