Techniques of  Deprivation Studies
 


 
 

Arousal Technique

    The earliest attempts at depriving an animal of sleep was to use electrical vestibular, or tactile stimulation to awake the animal whenever it entered the forbidden sleep stage (as identified by electrophysiological indices).  Not only were these methods extremely stressful to the animals, they also eventually became incapable of preventing target sleep stages as the experiment proceeded.

Watertank (Platform) Technique

     In this technique, the animal sits on an inverted flowerpot surrounded by water.  They can obtain slow wave sleep by sitting or crouching.  However, when they enter REM sleep their muscular tone diminishes and causes them to touch the water which wakes them up.  The advantages of this technique are that it is simple, and allows a large number off animals to be tested at one time. However, it is controversial with regard to behavioral consequences of the animals.  Additionally, it is questionable whether the control animals commonly used are appropriate.

Pendulum Technique

    The apparatus for this technique consists of a swing with room for three rats in individual cages.  The cages are slowly and continuously moved back and forth.  This produces imbalance in the animals and they walk downwards to the other side of the cage to prevent imbalance.  Sleep data taken during the deprivation period consistently indicate that percentage of time spent in REM sleep is negligible, making its efficacy parallel with the watertank technique.  Perhaps one advantage of the technique is that the REM sleep deprived state of the pendulum is taken into account by gradually increasing the speed of the pendulum, whereas the watertank technique is not modifiable.

    A newer version of the platform technique is the multiple platform technique that was designed so that the rat had less restriction in movement, and thus less stress.  In this technique multiple platforms are placed in one watertank and the animals can move from one to than other.


Platform vs. Pendulum

    Questions about the validity of these techniques stem mostly from the findings that different REM sleep deprivation techniques result in different effects of subsequent behavior (although this is not attributed to amount of REM sleep deprivation).   Additionally, it has suggested that the platform techniques may induce higher levels of non-specific stress in addition to deprivation.  In such experiments stress can be measured by ulcers in the stomach, weight loss, changes in the thymus and adrenal gland.  One factor may be the number of arousals which is greater in the pendulum than in the platform situation.  These “pendulum-platform” problems have yet to be solved.


Disk Technique

    This technique takes advantage of rats’ aversion to water.  Plastic cages house the experimental rat and its yoked control.  A plastic disk protrudes under each cage to provide a partial floor with approximately the same areas as a home cage.  The center of the disk is between the cages.  A tray of 2-3 cm deep water extends beyond the walls of the cage and beneath the side of the disk.  Rats are permitted to move freely throughout the cage but are connected to a recording cable.  This cable passes brain wave information to a polygraph in another room for continuous ink recording.  When the recording indicates that the rat has entered a particular sleep stage a microcomputer will rotate the disk at a rate of 3.33 revolutions/minute.  This movement forces the rats to awaken and walk in the direction opposite to disk rotation to avoid being carried into the water.  The rotation ends when the signals indicate that the rat has been awake for 6 seconds.  In total sleep deprivation studies, the disk will be rotated when signals indicate PS, low amplitude sleep, or high amplitude sleep.  In REM sleep deprivation studies, the disk will be rotated for REM sleep only.

     The experimental rat has a yoked control in the adjacent cage.  Whenever the experimental rat starts to sleep the disk is rotated and forces both rats to wake up and walk in the opposite direction.  Thus both rats receive the same physical stimulation but that is targeted to awaken the sleeping experimental rat.  In most studies the yoked controls remain healthy throughout the testing period (in contrast, the experimental rat usually dies). Unfortunately, yoked controls were imperfect because as sleep deprivation continued, deprived rats entered the pan more often than their yoked controls, because they often were too tired to get up!  Additionally, most immersions of yoked and experimental are partial but later on in deprivation rats would enter the water fully or when they failed to wake up or were unable to walk seemingly because of weakness. However, overall, immersion is relatively infrequent, with immersion rats usually being less than 2% for most experiments.

    Some studies also use an immersion control.  The frequency and duration of partial immersion and full immersion is determined in the experimental rat.  Immersion control rats are then maintained on similar disks that could be lowered into the pan. These rats are automatically immersed to match the frequency of immersion in the experimental animals.


Duration of Sleep Deprivation Studies

Most sleep deprivation studies last many days.  The need for a prolonged period of study stems from the possibility that response to short-term sleep loss may be the result of an attempt to defend against deprivation-induced impairment by restoring sleep, rather than the relevant impairments themselves.   For example, performance is frequently impaired in the form of lapses which may be the intrusion of incipient sleep processes. Additionally, because sleep takes time away from other adaptive functions, it must serve a biological function and the experimenter may need to enforce deprivation for a long time to reveal deprivation-induced deficits. In most sleep deprivation experiments, animals are sacrificed within a day or two of probable death in order to obtain viable tissue samples.  This usually is within 16-54 days for paradoxical sleep deprivation, 11-32 days for total sleep deprivation, and 22-66 days for NREM sleep deprivation.  Imminent death is judged by the following symptom

Severe debilitated appearance
Marked decline in maximal food intake
Severe ataxia or weakness
Decline in body temperature
Swelling of the paws
Loss of EEG amplitude
 
 

Advantages of this Technique

When we prolonged deprivation using intense, continuos and /or noxious stimulation it is difficult to say whether the impairments are from the sleep loss OR from stress and fatigue induced by the stimuli used to maintain wakefulness.  The solution to this problem was to maintain deprivation by benign stimulation that could be delivered to control subjects as well, without significantly affecting their sleep.

In comparison, the advantages of the disk method are numerous:
It is capable of preventing target sleep stages until death (although most experiments don’t go this far).
Only mild stimulation is used to awaken the rat
Stimulation only occurs when indicated by physiological recording (it is not continuous)
Delivery of the same stimulus to a yoked control
Requires only a simple, nontaxing response (awakening and walking a few steps) to stop stimulation.
 
 

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