Group Title: reliability of a depth of sleep measure and the effects of flurazepam, pentobarbital, and caffeine on depth of sleep
Title: The reliability of a depth of sleep measure and the effects of flurazepam, pentobarbital, and caffeine on depth of sleep
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Title: The reliability of a depth of sleep measure and the effects of flurazepam, pentobarbital, and caffeine on depth of sleep
Physical Description: vii, 106 leaves : ill. ; 28 cm.
Language: English
Creator: Bonnet, Michael Herbert
Copyright Date: 1977
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Subject: Barbiturates   ( lcsh )
Caffeine   ( lcsh )
Sleep   ( lcsh )
Psychology thesis Ph. D   ( lcsh )
Dissertations, Academic -- Psychology -- UF   ( lcsh )
Genre: bibliography   ( marcgt )
non-fiction   ( marcgt )
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Statement of Responsibility: by Michael H. Bonnet.
Thesis: Thesis--University of Florida.
Bibliography: Bibliography: leaves 100-105.
General Note: Typescript.
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Bibliographic ID: UF00098653
Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: alephbibnum - 000186418
oclc - 03372603
notis - AAV3008

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1


THE RELIABILITY OF A DEPTH OF SLEEP MEASURE
AND THE EFFECTS OF FLURAZEPAM, PENTOBARBITAL,
AND CAFFEINE ON DEPTH OF SLEEP










By

MICHIAEL H BONNET


A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL
OF THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY









UNIVERSITY OF FLORIDA


1977





ACKNOWLEDGMENTS


The author wishes to thank the members of his

committee, Dr. Keith Berg, Dr. C. Michael Levy, Dr. William

Yost and Dr. Steven Zornetzer for their time, interest, and

guidance not only in this project but throughout his

graduate career. Also the author must thank Dr. Doris

Chernik and Hoffman La Roche Company for their academic

and monetary interest in funding this project.

It is a humbling experience to look at the effort

others have invested in oneself. And the small return is

often shameful. A very long list of people appears to whom

the author owes gratitude for showing him what an educator,

a psychologist, and a friend could be. To parents and

friends and, perhaps most of all, to Dr. Wilse B. Webb,

the present work is dedicated as a very small interest

payment on a very large debt. As the son is the father to

the man, so the gifts many have given are expressed herein

and so shall they be expressed in any work of value which

this author produces.





TABLE OF CONTENTS


Page

. ii


. iv


. .v


. vi


. .1


. 23
. 23
. 24
. 27


. 40
. 40
. 41
. 43


. 61


. 76


ACKNOWLEDGMENTS ......


LIST OF TABLES .......


LIST OF FIGURES ......


ABSTRACT .. .. .. .


INTRODUCTION ........


EXPERIMENT 1 ........

Hypotheses ......
Method . . . .
Results and Discussion


EXPERIMENT 2 ........

Hypotheses ......
Method . . . .
Results . . . .


DISCUSSION .........


SUMMARY . . . . .


APPENDICES


A. THE: EFFECT OF FLURAZEPAM, PENTOBARBITAL,
AND CAFFEINE ON LATENCY TO SLEEP ONSET
ACROSS THE NIGHT ...........


B. THE EFFECT OF DRUGS ON TWO SUBJECTIVE
REPORT MEASURES. ...........


C. THE, RELATION OF DEPTH OF SLEEP TO
SUBJECTIVELY REPORTED SLEEP PARAMETERS


REFERENCE NOTES ... .............


REFERENCES. .. .. ..... .....


BIOGRAPHICAL SKETCH .... .........





LIST OF TABLES


Table Page

1 Step 1, 2, 3, and 4 Threshold Reliability
ANOVA's for Arousal Threshold .. .. .. 29

2 Step 4 Waking Threshold Reliability ANOVA . 30

3 Replication by Drug Condition by Trial by
Subject ANOVA for Flurazepam, Pentobarbital
and Placebo from "Pure" Sleep Data . ... 45

4 Replication by Drug Condition by Trial by
Subject ANOVA for Flurazepam, Pentobarbital
and Placebo from Waking Threshold Data . .. 46

5 Replication by Drug Condition by Trial by
Subject ANOVA for Flurazepam, Pentobarbital
and Placebo from Combined Waking and Sleeping
Data .. .. .. ... .. . .... . 47

6 Drug Condition by Trial by Subject ANOVA
for Flurazepam, Pentobarbital, Caffeine,
and Placebo from "Pure" Sleep Data . ... 48

7 Drug Condition by Trial by Subject ANOVA
for Flurazepam, Pentobarbital, Caffeine,
and Placebo from Waking Threshold Data . .. 50

8 Drug Condition by Trial by Subject ANOVA
for Flurazepam, Pentobarbital, Caffeine,
and Placebo from Combined Waking and
Sleeping Data .. .. .. ... ... 51

9 Drug Condition by Trial by Subject ANOVA
for the Disassociation of EEG Breakup and
Verbalization as a Function of Tone
Intensity . .... .. .. .. .. .. 60

10 Drug Condition by Time of Night by Subject
ANOVA for Sleep Latency Rank after an
Awakening .. .... . .... .. .. 80





LIST OF FIGURES


Figure Page

1 Within and Between Subject variance in
Arousal Threshold of Two Subjects over
Three Baseline Nights .. .. .. .. .. 34

2 Three-night Average of Arousal Threshold
(Corrected for Waking Threshold) for the Six
Subjects of Experiment 1 . . .. . 36

3 Average Depth of "Pure" Sleep Across the
Night after Administration of Flurazepam,
Pentobarbital, Caffeine, or Placebo .. .. 52

4 Average Threshold of Awake Subjects Across
the Night after Administration of Flurazepam,
Pentobarbital, Caffeine, or Placebo .. .. 53

5 Average Combined (Waking and Sleep) Threshold
Across the Night after Administration of
Flurazepam, Pentobarbital, Caffeine, or
Placebo .. .. .. .. ... .. .. 54

6 Sleep Latency after an Awakening Across the
Night in Flurazepam, Pentobarbital, Caffeine,
and Placebo Conditions . ... ... .. 83
















Abstract of Dissertation Presented to the
Graduate Council of the University of Florida
in Partial Fulfillment of the Requirements
for the Degree of Doctor of Philosophy


THE RELIABILITY OF- A DEPTH OF SLEEP MEASURE
AND THE EFFECTS OF FLUR~AZEPAM, PENTOBARBITAL,
AND CAFFEINE ON DEPTH OF SLEEP

By

Michael H. Bonnet

June 1977

Chairman: Dr. Wilse B. Webb
Major Department: Psychology

In the initial experiment a reliability estimate of

the presently used depth of sleep measure was ascertained,

both for the raw threshold data and for data which resulted

from three transformations to add control. For six trials

(an average night) in six subjects, the reliability estimate

for the raw data was .90 and for the controlled data was

.95.

The sensitivity of the threshold measure was then

tested in a study involving flurazepam, pentobarbital,

caffeine, and placebo conditions. Thresholds were measured

from a standardly defined segment of Stage 2 sleep for six

subjects for eight nights. Flurazepam and pentobarbital

were seen to increase thresholds, both in a pure sleep

measure and in awake thresholds, in a time course fashion





across the night. Trend analyses indicated that flurazepam

was both a faster-acting and a longer-lasting drug than

pentobarbital, but the main effects of both drugs on sleep

thresholds were seen in the first 3 1/2 hours of sleep.

Caffeine administration resulted in lower thresholds, as

a concomitant largely of lower awake thresholds, than the

placebo or the other drugs. Such evidence favors the

contention that depth of sleep is, at least in some aspects,

a different measure of sleep behavior from the EEG because

depth of sleep has been shown to vary within EEG sleep

stage. However, hypotheses designed to show the operation

of a dual arousal system which could explain both sleep

depth and EEG findings by showing differential arousal

characteristics for the barbiturate versus the benzo-

diazepine were not upheld. A central mechanism for sleep

depth which was independent but related to EEG controlling

mechanisms was implied by the findings but not identified.

Concurrent threshold results included evidence that

the awakening threshold is inversely related to the time

it took subjects to fall asleep again, and thus, that the

latency measure could also serve as an index of drug

activity. Both the latency information and the threshold

time course information were upheld to some extent by

subjective measures collected the following morning.

Substantial agreement among the three data bases was con-

sidered a validation of all three measures of the sleep

process.





INTRODUCTION


Where no part of the soul remained
behind, concealed in the limbs, as
fire remains concealed when buried
under much ash, whence would sense
be suddenly rekindled through the
limbs, as flame can spring up from
hidden fire?
Lucretius


The threshold of response is basic to all of percep-

tion, discrimination, and higher psychological functions.

As such, threshold maintains a predominant role in all of

behavior. It is perhaps fitting that the earliest studies

of sleep sought to determine awakening thresholds through-

out the night as their definition of the sleep process.

Elegant threshold curves were produced by De Sanctis and

Neyroz (1902), and others, and their data closely approx-

imated later electroencephalographic (EEG) pictures of the

time course of the sleep process. Threshold measures of

sleep were common for about 70 years, although very few

studies were done until the use of EEG became commonplace.

The appeal of the threshold studies was simply that behav-

ioral responsivity as a function of signal intensity is an

intuitively obvious way to approach sleep and one that

seems fairly simple.









There were some good reasons for the switch to EEG as

a measure, though. There are many serious problems

involved in sleep threshold experiments. Results from

different sensory modalities differ and may not be directly

comparable. Different types of required response result in

different data. Effects of instruction, expectancy, and

subject motivation can drastically alter results. The

manner of signal presentation affects measurement. The

number and order (Sokolov, 1963) of stimulus presentation

can affect threshold both in waking and in sleeping

subjects. Some of these problems as they apply to threshold

measurements during sleep have been briefly noted by Webb

and Agnew (1969), but it must be stressed that these

problems apply not only during sleep but also in waking

subjects. The further problem of an interaction of

sleep/waking with any of the above factors must also be of

interest.

As a part of the problem of threshold measures in

general, "the depth of sleep, like its quality, is an

elusive characteristic" (Kleitman in the 1939 edition of

his book). Several investigators have vainly sought to

untangle the complexities of the problem of sleep depth.

Four major problems have obscured a clear understanding of

depth of sleep. First is the disassociation of autonomic

measures during sleep. Heart rate, respiration, body

temperature, and the wYhole spectrum of autonomic

responses do not simply relate to sleep depth (Kleitman,









1939; Rechtschaffen, Hauri, & Zeitlin, 1966; Williams,

1967), nor do they relate to each other during sleep. The

implication is that if any one psychological measure is

used as the indicant of sleep depth, it will not agree with

either awakening thresholds or other physiological measures.

The second problem involves subject responses. Threshold

studies are based upon a subject response. If the presen-

tation of a tone is followed by a button press response in

two subjects and one of those subjects wakes up and remem-

bers the whole incident the next morning while the other

shows no other response or memory for the event, what does

one conclude about the two subjects? They appear to have

equal and yet unequal thresholds at the same time. Both of

the first two problems are "what are you interested in"

problems. If one is interested in a general physiological

index, the answer does not exist, but if one asks a specific

question such as, "How loud a sound does it take for a

person to say, 'I am awake,'" such problems dissolve except

perhaps at a philosophical level. A third problem is

perhaps a blessing in disguise. It is the discovery and

popularity of the EEG as a measure. Since the more com-

plete delineation of the EEG sleep cycle in 1953, there has

been a tendency to use EEG as the criterion of depth of

sleep rather than in investigating depth of sleep. For

this reason few systematic -studies of sleep depth have been

done (Williams, 1967). The fourth problem is really a

function of EEG measures and depth and involves the lack of





agreement between human and animal studies of sleep depth.

This has limited the exploration of sleep depth.

The use of the EEG as a measure of sleep avoids

several of the rather spectacular problems associated with

sleep depth measures, but the EEG has several problems

of its own as a measure. The major problems of EEG deal

with its relation to actual, observable behavior. At one

level, for example, Johnson (1973), in a review of the

relation of sleep stages to subsequent performance behavior,

said the relation "remains a mystery" (p. 337). At another

level, a whole series of research has observed that the

relation of ongoing EEG to behavior is not causal but only

correlational. The result is that an experimental manipu-

lation can separate the two. As early as 1938, Davis,

Davis, and Thompson reported that subjects breathing gas

mixtures containing half as much oxygen as normal showed

delta activity while still being responsive to commands to

open or close their eyes or to write. Drugs produced

simi la r e ff ects For example, atropine produced slow waves

similar to delta in animals that were neither drowsy or

unresponsive (Bradley, 1958; Wikler, 1952, cited in Lacey,

1967). As such, if drugs disassociate EEG from behavior,

EEG would seem a very poor measure to use in describing the

effects of drugs because any effects seen might have little

relation to behavior. Feldman and Waller (1962) found that

animals with lesions in the reticular formation showed slow

wave cortical activity while behavioral activity was








present. Conversely, lesions in the posterior hypothalamus

resulted in animals behaviorally comatose who still showed

fast wave, "waking" brain activity in response to reticular

stimulation. Lacey in a 1967 review concludes that "elec-

trocortical arousal, autonomic arousal, and behavioral

arousal may be considered to be different forms of arousal,

each complex in itself" (p. 15). In short, EEG is never a

perfect correlate of behavior and occasionally is randomly

or even inversely related to behavior. Further, the dis-

association may be rather common. It may even be seen in

sleep deprivation in normal human subjects (Blake, Gerard,

& Kleitman, 1939).

There is one more disturbing problem with the use of

EEG as a measure. The Feldman and Waller study (1962)

hypothesized that while the reticular system mediated

cortical tone, the posterior hypothalmic region had a

functionally separate role in maintaining waking. If that

hypothesis were correct, and the data did support it, it

would mean that the simple use of EEG measures would over-

look an entire dimension of behavior--specifically that

mediated by the hypothalamic system.

Threshold measures, which form the basis for the label

"sleep depth" herein, traditionally have been oriented

toward the realm of physical behavior (here defined as

requiring a motor response). As such, the problem of the

degree of correlation with motor behavior is nonexistent.

Because of this the hypothalamic components proposed

by Feldman and Waller could perhaps best be measured by a









threshold type study. The problem is that a whole gamut of

learning effects and all the shortcomings of threshold

measures are also introduced. The potential experimenter

is faced with a mass of variables and interrelationships.

This is both good and bad. Because of this, researchers

in the area call for more studies and bemoan the fact that

depth of sleep is a useless variable. In 1967, Williams

rhetorically asked if the term "depth of sleep" should be

given up. He answered "no," because no sequence of experi-

ments controlling all the relevant variables had been done.

In a following discussion Snyder agreed with Williams in

these words (Note 5):

It has become kind of a cliche among workers in this
field that the concept of depth of sleep has little
meaning, yet I think that we all have a great deal
of conviction that in terms of common sense experience
is a very important idea. Perhaps if our science
cannot provide an operational definition of depth of
sleep, then this is the short-coming of our science.
(p. 314)

To complete the circuit of perhaps the three most knowl-

edgeable researchers in the area, Rechtschaffen et al.

(1966) said,

Awakening threshold has probably not received the
interest it deserves in its own right. .. Surely
such an adaptive mechanism represents the product of
"careful" natural selection and deserves attention
as a fundamental biological phenomenon. (p. 937)

A recent review (Bonnet, Note 1) cites about 150

references in the area of depth of sleep. A complete

review is clearly inappropriate to the present work with

three broad and important exceptions--the reliability of





the measure, the present use and usefulness of the measure,

and the application of the measure in new areas.

Central to the experimental use of any measure are

questions concerning the reliability and the validity of

that measure. While several sources have commented on

large between- and within-subject differences in threshold

during sleep, only one source (Note 10) has commented on

the reliability of such measures. In that study, as the

result of a complex awakening schema, six subjects were

classified as either light, medium, or deep sleepers.

Using the same schema on a second night, three were rated

the same and three shifted by one group. On these results,

reliability was deemed "sufficient." The lack of measure-

ment reliability data in a measure known to exhibit wide

variability is unfortunate because it limits conclusions on

the validity of findings. A major reason for the paucity

of data in this area is probably the fact that threshold

measures, in general, have been taken to present consider-

able face and content validity. Validity, of course, is

not reliability, and a truly valid waking measure is not

necessarily a reliable or valid measure of sleep.

Even with the assumption that depth of sleep measures

are both reliable and valid, one must question their value.

Are they responsive to parameters that sleep as a behavior

should be responsive to? Can information not already

obtained from EEG studies be obtained by the use of such

measures? Although systematic studies have- not been done





to answer these questions, studies relevant to both have

been done. A large body of information has grown concerning

information processing during sleep (see, for example,

Williams, 1973). A number of studies have been reported

spun off from the classic Oswald, Taylor, and Treisman

(1960) study, in which both EEG and arousal probabilities

were greater to a subject's name than to another name or

the name played backwards. Sleep depth obviously varies

with signal relevance. Either as a part or independently

of signal relevance, subject motivation, in terms of pay-

ment for hearing a signal versus nonpayment, has also been

shown to have a large effect on responsivity during sleep

(Wilson & Zung, 1966; Zung & Wilson, 1961). On the other

hand, a study designed to find the effects of subject

motivation, again in terms of payment, on the EEG sleep

stage distribution across the night (Bonnet & Webb, 1976)

found only marginal changes in EEG patterns.

Another variable which sensibly should affect the

sleep process is the length of prior wakefulness. A large

volume of work (see, for example, Agnew & Webb, 1971;

Webb & Agnew, 1971) has documented the EEG effects of prior

wakefulness. One study (Williams, Hammack, Daly, Dement,

& Lubin, 1964) has examined the effects of prior wakeful-

ness on behavioral threshold. Very briefly, it was found

that 64 hours of sleep deprivation, in addition to changing

the EEG sleep distribution, also lowered the number of

responses to tones within sleep stage. While the response





percentage in Stage 2 sleep was 24% before deprivation, it

was only 4% after deprivation. As a comparison, the

response level in Stage 4 before deprivation (9%) was a

good deal greater than responsiveness in Stage 2 after

deprivation. This study has the interesting conceptual

addition that sleep depth can vary within sleep stage and

could therefore represent the operation of different

systems in the sleep response.

The characteristic EEG distribution across the night

was documented in detail as early as 1964 (Williams, Agnew,

& Webb). Five sources (Goodenough, Lewis, Shapiro, &

Sleser, 1965; Keefe, Johnson, & Hunter, 1971; Rechtschaffen

et al., 1966; Shapiro, Goodenough, & Gryler, 1963; Watson

& Rechtschaffen, 1969; Zimmerman, 1970) point to a charac-

teristic pattern of depth of sleep across the night which

operates not only with sleep stage but also within sleep

stage. The major characteristic of that pattern is a

lightening of sleep within sleep stage as the night progres-

ses.

Circadian effects on the sleep process as measured

by EEG have been reported (see, for example, Agnew &

Webb, 1973; Webb & Agnew, 1971; Webb, Agnew & Williams,

1971). One study, reported by abstract only (Note 3),

has examined the effects of sleep placement on depth

of sleep. Corvalan and Hayden (Note 3) determined awak-

ening thresholds for subjects in sleep periods beginning

at 10:00 p.m., 8:15 a.m., 9:15 a.m., 10:15 a.m., and










noon. A wide range of threshold values was reported in

each stage of sleep, and it appeared that the values from

REM and Stage 4 depended upon real time. The highest

values were recorded between 10:00 p.m. and 8:00 a.m.

Corvalan (1969) reports reaction time to a constant level

stimulus during sleep across day and night. Only reaction

time during REM appeared time locked, and it was seen to

decrease with absolute time across the 24 hours. From the

brief information, there appear to be many problems with

these experiments. There was unequal distribution of time

of sleep period with none occurring in the evening, and

there appeared to be no attempt to control for effects of

prior wakefulness. In regard to Stage 4, results are

inconsistent--one method found time dependency and one did

not. The results on REM agree with those studies examining

sleep depth during the normal sleep time. However, evi-

dence does indicate the presence of some circadian effects.

Two other variables, drugs and age, have been found to

have profound effects on EEG patterns. However, neither of

the variables has been examined for sleep depth character-

istics. There is a need for further information in both

areas, not only because such information will establish

further the covariation of EEG and threshold measures, but

also because there is a tremendous potential for both

applied and theoretical data in both areas. In the present

studies, the area of drug effects on depth of sleep will be

examined more closely.





There have been two common approaches in the study of

the effectiveness of drugs in human sleep. The earliest

very simply involved administering the drug and asking the

subject in the morning how he had slept. Such procedures,

of course, allowed several types of bias, and the relation-

ship between sleep depth and such ratings is unknown. Jick

(1969) reviewed this data and found it difficult to differ-

entiate hypnotics used within this methodology.

The second approach, which is at present the common

approach, was to use the EEG as a measure. Several recent

reviews are readily available including Oswald (1968, 1969,

1973, 1974), Hartman (1969, 1973), and Winters (1969). The

approach of all the reviews and of the studies reviewed has

been similar. Following drug administration, EEG measures

of sleep latency, sleep stages, sleep stage shifts, and

sleep length were recorded and compared. A very recent and

interesting application of the EEG in the examination of

ongoing brain activity to chart drug effects has been the

development of what Itil has called "sleep prints" (1976).

Such prints chart occurrence of various wave frequencies

in the EEG more completely than is seen in sleep staging.

Flurazepam, for example, results in a sharp decrease (over

placebo) in wave frequency seen about two hours after sleep

onset followed by an increase (above placebo) in frequen-

cies, peaking at about four hours after sleep onset and

staying above placebo for the remainder of the night. The

meaning of these changes, however, is not clear. Although









shifts in stage distributions have been associated with

"deeper" or "lighter" sleep as a result of the general

relation between sleep stagh and sleep depth, the variable

of sleep depth within sleep stage was never mentioned. A

rather thorough review of the literature revealed a rather

alarming paucity of studies (Bonnet, Note 1).

The single most applicable drug study involving sleep

depth in the published literature was performed by Lindsley

in 1957. He was interested in response rate (a button

push) to reduce tone intensity as a result of several sleep

conditions including sleep deprivation and a dose of

seconal. Seconal is a short-lasting barbituate which

usually induces sleep in 15-20 minutes. Lindsley's data

showed normal response rate during about a 15-minute sleep

latency (slightly shorter than the two subjects' normal

latency). Sleep onset was followed by approximately 4

hours of virtual nonresponse mirroring almost exactly the

nonresponse characteristic of 38 hours of sleep deprivation.

At this point responding began and increased. With

responses during sleep latency subtracted, about 600

responses were made during the control night, about 440 on

the drug night, and about 300 on the night following sleep

deprivation. It is obvious that the experimental conditions

had an effect, but, of course, it cannot be determined if

those effects were due to increased amounts of stage 4 or

increased difficulty of responding within stages or both.





Sedatives have been found not to alter brief latency

evoked potentials but to modify the threshold and waveform

of longer latency responses (Davis, 1973; Price & Gold-

stein, 1966). However, with the exception of comments by

Monninghof and Presbergen in 1883 that consumption of a

large amount of alcohol made subjects less sensitive to

stimulation in earlier parts of the night, sleep depth

during the night has simply not been examined except

circuitously. Mullin, Kleitman, and Cooperman (1933)

examined the effects of alcohol and caffeine on motility

during sleep. The results indicated that alcohol consump-

tion caused a distinct reduction in body movement during

the first half of the night and a possible reduction in

movement for the entire night and that caffeine produced a

marked increase in motility during sleep. Mullin, Kleit-

man, and Cooperman (1937) found that sleep depth was

related to the length of time following a body movement.

Such would imply that alcohol deepened sleep while caffeine

lightened it. Still these findings could have been the

result of shifts in sleep stage distribution as easily as

a threshold shift within sleep stages.

In a pair of more recent studies (Itil, Saletu, Marasa,

& Mucciardi, 1972; Itil, Saletu, & Marasa, 1974) subjects

were awakened at 5:00 a.m. by a tone series (1000 Hz, I

second in duration) beginning at 40 dB (reference not

given) and increased in 10 dB steps to 80 dB. The 5:00 a.m.

awakening was not controlled for sleep stage and occurred





seven hours after drug administration and attempted sleep

onset time. Flurazepam and U-31,889, which is a triazolo-

benzodiazepine derivative, were tested against placebo in

the 1972 study. At 5:00 a.m., thresholds were significantly

higher to the highest dose of U-31,889 (2 mg) than to any

other drug or placebo condition. The major problem with

the study is that both flurazepam and U-31,889 are classed

as short-acting drugs with time courses usually estimated

at less than 7 hours. As such one would predict the null

hypothesis in all conditions at a test 7 hours after admin-

istration. The finding with the highest dose of U-31,889

would, however, additionally raise the question that not

only depth of sleep but also drug time course might

hypothetically vary with drug dosage. However, the poten-

tial results of this study are further confounded by the

fact that no control was exercised on awakening parameters

(with the exception of time). It is a fact that most drugs

alter sleep stage characteristics in complex fashions so

that the probability of sleep stage occurrence in the

reported experiment differed with the drug given. There

is, therefore, a nonrandom probability of occurrence of any

sleep stage at 5:00 a.m. This could account for the

reported differences (or cover more extreme differences)

even though threshold differences between Stage 1-REM and

Stage 2 appear minimal (see Note 1). Further, it is con-

ceivable that some condition might predispose subjects to

have increased awakenings or to be awake naturally around









5:00 a.m. (i.e., early morning). Because thresholds are

lower shortly after an awakening (Mullin, Kleitman, &

Cooperman, 1937), this could also confound results. The

1974 study contains the same methodology and faults. In

it, methaqualone, triazolam, and flurazepam were tested

against placebo, and thresholds were significantly greater

than placebo with triazolam and flurazepam at 5:00 a.m.

In a third study reported in Williams and Karacan (1976)

no difference from placebo was found at the 5:00 a.m.

awakening for diazepam or clorazepate dipotassium.

Some information on drug effects is also available

from other sources. As a part of some methaqualone studies

done in the Florida Sleep Laboratories (Note 9), Rorer

provided some brief results of studies they had done on

their product (Quaalude). They reported that a dose of

10 mg/kg usually increased voltage of sciatic nerve stimu-

lation necessary for EEG arousal in the cat by 100%. A

dose of 5 mg/kg elevated threshold by 70%. Sleep stage

tested wyas not specified or cited as controlled, and the

nonrandom variations earlier discussed probably did exist.

Natural within-sleep threshold shifts may or may not have

played a factor. However, if those variables could be

considered random, the study provided evidence for a drug

threshold shift in the cat.

Other animal studies, recording from various sites

ranging from the limbic system to the reticular formation,

have found increases of threshold to various arousal









responses (Gogolak & Pillat, 1965; Randall, Schallek,

Scheckel, Stefko, Banziger, Pool & Moe, 1969; Schallek &

Kuehn, 1965; among others).- The usual stimulus has been

stimulation in the reticular formation. Selective thresh-

old increase at limbic versus reticular sites in these

studies has been taken as evidence of site of drug action,

a point which will be discussed later. It should be

mentioned that these findings are not universal. Lanoir

and Killam (1968) found that their cats did not even go to

sleep after administration of valium or mogadon. However,

for present purposes, an in-depth review of this data

beyond the statement that threshold increases have been in

general found is perhaps not strictly relevant for the

following reasons. In all of these studies the point of

stimulation and the point of measurement have usually been

deep within the brain, and the relation to "real world"

stimuli or behavioral responses is uncertain. Also, the

initial criticisms of the results provided by Rorer hold

generally. The largest problem, however, is that it has

been well established that sleep depth in animals does not

correspond well with sleep depth in humans (Jouvet, 1961;

Williams, 1967; or see Bonnet, Note 1, for review) as it

relates to EEG sleep stage. The strong implication is that

the sleep system in man is different from that of the rat

or cat. Human data, conceivably, relates only tangentially

to the animal data, and the present concern is primarily

with the human.









The state of the area of human research on thresholds

after drug administration is summarized by Itil in a chap-

ter published in late 1976:

Despite the fact that awakening threshold seems to
be one of the most important parameters for deter-
mination of the quality of sleep, we were unable to
find any recent literature regarding that measure-
ment. (p. 236)

As a part of a pilot study in 1975, two human subjects

have been run in our laboratory. Subjects were given a

standard 300 mg tablet of Quaalude 15 minutes before

retiring on the drug night. Subjects had been previously

adapted to the laboratory. Five-second segments of audi-

tory stimuli were presented in an ascending method of

limits design which terminated when a button push response

was made. Stimuli were presented only in Stage 2 sleep,

and 5-7 arousals were made per night. Data from each

subject were plotted with data points connected by straight

lines. Interpolated threshold at eight times across the

night was averaged between subjects. The results were

clear. (a) Threshold drops on both the drug night and

control night were seen over the sleep period within

Stage 2. The finding replicates Rechtschaffen et al.

(1966), Watson and Rechtschaffen (1969), Zimmerman (1970),

and Keefe, Johnson, and Hunter (1971) among others.

(b) The Quaalude appeared to stably increase Stage 2

threshold by about 8 dB throughout most of the night.

Because neither of these results could have been predicted

from simple monitoring of the EEG, it seems that a more





encompassing view of sleep as a process is necessary. One

possible approach is the dual arousal system approach

advanced by Routtenberg (1966, 1968) and modified to apply

specifically to studies of responsivity during sleep by

Bonnet (Note 1). Very briefly, the models proposed that

two arousal systems mutually interact and that responsivity

is a function of activity in both. Arousal System I is the

classic reticular system, which greatly influences the

cortical EEG. Arousal System II is a limbic midbrain

system involved primarily with incentive and less completely

represented in cortical EEG (Feldman & Waller, 1962). This

implies that threshold shifts mediated by Arousal System II

activity may not be adequately represented in ongoing EEG.

Such is a conceivable explanation of why some insomniacs

complain of getting little or no sleep while displaying

normal EEG sleep activity. This proposition (and others)

could be tested only by use of sleep depth measures.

In terms of drug effects in the two hypothesized

systems, there are a large number of animal studies at two

levels of examination. At the more gross level, which can

be taken to mean results by experimenters recording brain

rhythm activity from various brain structures after drug

administration, evidence suggests that (1) barbiturates act

at the level of the reticular formation (Kido & Yamamoto,

1962; Schallek & Kuehn, 1965; Schallek, Kuehn, & Jew,

1962); and (2) that minor tranquilizers work by inhibiting

limbic structures (Schallek & Kuehn, 1965; Schallek et al.,





1962; Schallek & Thomas, 1971; Steiner & Hummel, 1968).

Olds & Olds (1969) concluded,

There now appears to be general agreement that there
are drug receptors in the mesencephalic reticular
formation which are directly acted upon by barbi-
turates before action on other structures occurs,
and that such action is largely responsible for
the behavioral and EEG effects noted with these
compounds.

On the other hand, the benzodiazepines appear to have a

"selective action" on hippocampal neurons (p. 100).

However, Fuxe and his coworkers, working at the neuro-

transmitter level, have identified several transmitter

pathways in the rat brain but have had little luck in

differentiating barbiturate and benzodiazepine action in

at least eight studies in the past eleven years (see Fuxe,

Hokfelt, & Ungerstedt, 1970; or Lidbrink, Corrodi, & Fuxe,

1974).

There is at present no easy way to combine these two

bases of data and additionally no promise that neuro-

physiology in the rat is similar to neurophysiology in

the human. It is the human case, of course, in which one

cannot implant electrodes or lesion, that is of primary

interest. Gross effects are all that one could hope to

find.

Routtenberg (1968) reviewed a finding by Kornetsky and

Bain (1965) that pentobarbital and chlorpromazine adminis-

tration altered waking responses to a choice task in

animals. Pentobarbital increased errors of commission

while chlorpromazine increased errors of omission. This





was suggestive to Routtenberg that the locus of the action

of the drugs might be different and the chlorpromazine

might act to depress Arousal System II. Such would be a

logical place of action for a tranquilizer. Because

cortical synchrony is primarily controlled by Arousal

System I (Routtenberg, 1966), barbiturate effects would

easily be seen in the EEG (Olds & Olds, 1969). Arousal

System II depression would be seen only to the extent that

it affected Arousal System I. If overall behavioral

responsiveness (i.e., depth of sleep) were modified by

levels in both systems, drugs acting on Arousal System II

might increase sleep depth without significantly altering

the recorded EEG. Further, it is conceivable that arousals

themselves differ in EEG terms depending upon the locus of

action of the drug involved.

Obviously, the arousal mechanism is extremely complex.

Chances of being able to differentiate drug activity by EEG

arousal characteristics are possibly slim but conceivable

within the model proposed and on the basis of the site of

action data.

In addition to these theoretical points it is held

that the measurement of thresholds during the sleep process

would give several sorts of useful applied data. Perhaps

the single most important contribution is the ability of

threshold measures to track a behavioral time course of

drug effects. As such drugs may be differentiated by

different time course attributes and classed according to









behavioral speed of action, length of action, and amount of

impairment. Amount of behavioral impairment might also

vary with dose. The practical question here is what

happens to a patient treated with a specific drug if a fire

alarm goes off during the night (etc.). Can he be awakened?

Can he stay awake? If awakened, can he go back to sleep if

he wants? How quickly can he go back to sleep? Or, what

should be given to a patient with noisy neighbors? With

sufficient time course information drug and dose could be

matched to result in maximum depth at time of maximum

noise. The point is simple. There is only one way to

determine the effects on behavior of any drug as it inter-

acts with the sleep process. That way is to sample behav-

ior at appropriate times during the drug activity. The

first step must explicate the effect of drugs on sensory

mediated responses.

The present state of knowledge in threshold studies

and drug studies indicates that a dual experiment is

necessary to begin to behaviorally document drug activity

during the sleep period. As a first step and initial

experiment the reliability of a threshold measure must be

established. Given measurement reliability, an attempt to

control the threshold variable with various drugs can be

made.

Finally, it must be recognized that an arousal from

sleep is dependent upon two factors. An awakening .thresh-

old is dependent upon the threshold of the subject when





22


awake plus the amount of threshold impairment caused by the

sleep process. The behavior of an awake subject will

always be referred to as waking behavior or waking thresh-

old. The sum score will be referred to as awakening

threshold or arousal threshold. Awakening threshold minus
waking threshold equals "pure" sle dph The waking

threshold and pure depth of sleep measures may operate

independently.





EXPERIMENT 1


Hypotheses


In terms of the reliability determinations, it was

hypothesized that there would be substantial measurement

reliability but that that reliability would be dependent

upon several controlled variables. (1) Because auditory

thresholds are routinely taken on many people, it was

hypothesized that waking threshold measures would be

extremely reliable and would form a ceiling that would not

be surpassed by the sleep measures. Differences between

the waking and sleeping reliability figures might be

attributable to the sleep process itself. (2) In the sleep

data four graded sets of data were examined with the

hypothesis that each "grade" or extra step of control would

increase measurement reliability. Step 1 was raw awakening

threshold data. Step 2 was Step 1 data with the corre-

sponding waking threshold subtracted ("pure" sleep depth

data). This controlled for movement of an earphone in the

ear and circadian effects as well as removing any other

effects of waking threshold. Step 3 was a transformation

of the Step 2 data. All data were plotted as a function of

time of night when the threshold determination was made,

and the points were connected by straight lines. The night

was divided into seven equal time periods by six points.









Six points were used because an average of more than six

but less than seven data points could be collected per sub-

ject per night. The interpolated value for each subject

was read from the graph at each point. This step was taken

so that an adjustment could be made to account for time of

night effects as well as individual patterns in sleep depth

across the night. The Step 4 analysis was to eliminate

data from the first laboratory night, which is often

uncharacteristic.

A final aspect of reliability is time. Subject nights

in Experiment I were three to five nights apart. The

effects of a longer measurement interval (two months) will

be reported as a part of Experiment 2.


Method


Six male subjects aged 21 to 23 were selected from

responses to an ad in the student newspaper for sleep sub-

jects. They were screened with the Florida Sleep Inventory

to have normal sleep habits including an approximate

11:30 p.m. bedtime and eight-hour sleep length. Subjects

were paid $15 per night for their services. Each subject

was given a series of tests including the Spielberger Trait

Anxiety Scale, the depression scale of the MMPI, the Adjec-

tive Check List, and a selected medical history. In addi-

tion, subjects were chosen who produced alpha and reported

minimal drug use.

Thresholds were determined by a modified Tracor RA 214

Rudmose screening audiometer. The audiometer was built to








ANSI 1969 specifications and calibrated by an audio

specialist at the ear insert output before experiments were

begun. Readings reported in this study are SPL (reference

.0002 dynes/cm2) levels. Modifications of the audiometer

included the addition of a resistor such that signal inten-

sities measured at hearing-aid ear inserts could be meas-

ured between -10 and 102 dB SPL at 1000 Hz in 2 or 3 dB

steps. Signals were presented in a three-seconds on,

three-seconds off sequence with interpolated catch trials.

Normal, waking thresholds generally are 7 + 15 dB (personal

communication from W. Yost), and all of the subjects were

in that normal range. All thresholds were obtained from

stepwise procedures, which with the exception of catch

trials, changed direction after a subject response change.

All subjects had half an hour of practice previous to any

experimental nights during which their "normal" thresholds

were determined.

Each subject spent five nights in the lab. Nights

were three to five days apart. Subjects reported to the

lab at 10:00 p.m., had electrodes attached so that record-

ings could be made from Fl-F7' P1 5, 03-OfzP (Oz z is

halfway between Oz and Pz) and an eye channel. A navel

body temperature probe was also attached. Subjects com-

pleted the Spielberger State Anxiety Scale and a day events

questionnaire before entering the sleep rooms at 11:00 p.m.

Subjects were allowed to insert the earphone into their

ear, and a response button was taped into their preferred

hand. Subjects were instructed to press the button









whenever they heard the tone and their responses, as well

as a signal marking stimulus onset and stimulus offset,

were fed through the EEG machine and written out as they

occurred. Any response which occurred during a stimulus

presentation was counted as correct. Responses during

catch trials and in between stimulus presentations (false

alarms) were minimal. On the rare instances of responding

on catch trials subjects were innocuously warned that there

might be catch trials so that they should try to listen for

the tone. From 11:00 to 11:30 p.m. subjects were allowed

to read or study while equipment was tested. During this

time, each subject's waking threshold was determined once

more. Lights were turned out at 11:30 p.m.

Subjects had practiced the threshold task and were

aware that their threshold would be tested between five and

eight times during the night. Each waking and awakening

threshold determination began with an ascending series and

ended when stable ascending values were obtained. Subjects

were instructed that when awakened during the night, they

were to say "I am awake" the first time they heard the tone

in addition to pushing the button in their hand each time

they heard the tone. When both responses were made (and a

waking EEG had appeared), a small night light was turned on

in the room to define the waking threshold determination

period, a period lasting about two minutes except when

subjects fell asleep during it. Waking threshold was





defined as "stable" when the value for three ascending

series was the same determined from a stepwise procedure.

On one of the five nights (night 3 for 2 subjects,

night 4 for 2 subjects and night 5 for 2 subjects),

subjects remained awake so that data on threshold varia-

bility across the night without sleep could be compared

with the data on arousal threshold and waking threshold on

sleep nights. On the other four nights, the first thresh-

old determination was made five minutes into the first

Stage 2 period. Thereafter, the following criteria were

imposed before a threshold determination was made: at

least 5 minutes into Stage 2; at least 30 minutes since the

last natural or experimentally produced awakening; at least

10 minutes since the last transitory body movement or

muscle artifact greater than 6 seconds; well-defined

Stage 2. Subjects were not awakened more than eight times

on any night.

Subjects were awakened finally at 7:30 a.m., and

apparatus was removed. Subjects filled out the Post-sleep

Inventory (Webb, Bonnet, & Blume, 1976) and were allowed to

leave the lab.


Results and Discussion


The major statistical tool used in estimating the

reliability of the threshold data was the method of intra-

class correlation (Guilford, 1954), an ANOVA procedure.

Between four and eight observations were collected on each









subject on each night., To keep equal numbers in each ANOVA

cell, ANOVAls for Step 1 and Step 2 comparisons were based

on the first four observations for each subject. Step 3

and Step 4 ANOVA's were based on the average number of data

points collected per night per subject (six). ANOVA's had

variance terms for trials across a night, nights, subjects,

error and interactions. Because degrees of freedom for all

interactions were greater than six and interaction F-values

were less than 2.00, the interaction sums of squares were

pooled with the error sums of squares to form one error

term (Hays, 1973). Degrees of freedom were similarly

pooled. Results of the Step 1 through Step 4 ANOVA's can

be seen in Table 1. The ANIOVA for the waking threshold

equivalent to Step 4 (without waking threshold subtracted)

can be seen in Table 2. The Pl values, expressing the

average strength of trial to trial relation of threshold

(approximating Pearson r) for a single trial, most graphi-

cally illustrated the effects of adding controls. Moving

from Step 1 through Step 4 respectively, the El values were

.61, .65, .80, and .86, and the error term was reduced on

each step. The waking value was .92. In terms of variance

accounted for in the sleep data, the Step 4 data accounts

for twice as much as the Step 1 data (74% vs. 37%) but is

still smaller than the 85% accounted for in the waking data.

Simultaneous with the present experiment, Johnson

(personal communication) collected some threshold data in

various stages of sleep as a part of a larger experiment.












Table 1. Step 1, 2, 3, and 4 Threshold
Arousal Threshold


Reliability ANOVA's for


Step 1 ANOVA (see text)


SS
208
1736
17311
11320


MSE
69
579
3462
135


F -for
Ss Effect
F =25.6


Ss
Intraclass
Correlation
216 = .96
'4 = .86
l= .61


source
TR
Nights
S
pooled error


Step 2 ANOVA


source
TR
Nights
S
pooled error


SS
138
1165
20479
11318


MSE
46
388
4096
135


F -for
Ss Effect
F = 30.3


Ss
Intraclass
Correlation
E16 = .97
24=.88
21= .65


Step 3 ANOVA


F -for
Ss Effect
F = 94.6


source
TR
Nights
S
pooled error


SS
609
1443
33662
9261


MSE
122
481
6732
71


Ss
Intraclass
Correlation

'6 = .96
rl = .80




Ss
Intraclass
Correlation
E18 = 99
?6=.97
rl = .86


Step 4 ANOVA


SS
272
558
26678
4489


MSE
54
279
5336
47


F -for
Ss Effect
F =113


source
TR
Nights
S
pooled error


S = subjects (These symbols are used on all tables.)
R = replication
DRUG = drug condition
TR = time trial across night





Table 2. Step 4 Waking Threshold Reliability ANOVA


Step 4 ANOVA


df SS M1SE F -for
5 53 10 Ss Effect
2 240 120 F = 209


Ss
Intraclass
Correlation
r = .99
218 9
26=.92


source
TR
Nights
S
pooled error


5 10049
95 911


2010









In what would correspond to a presented rl of .61 for a

single trial unadjusted arousal threshold in the present

data, his average correlation in Stage 2 was .36. Several

large methodological differences as well as data from two

small samples could account for the rather large discrep-

ancy.

In the present results, it was reported that reliabil-

ity was increased (to .65 for a single trial) by adjusting

the sleep threshold scores for the corresponding threshold

when awake. This effect, which accounted for movement of

the earphone in the ear canal and circadian threshold

shifts, appears to be real--i.e., not a function of the

simple effect of measure combination. Waking threshold was

not significantly correlated to sleep threshold between or

within subjects.

The second reported increase in reliability (from .65

to .80) presented is probably an artifact in the present

data. The interpolation method involved using a weighted

average of observations such that two observations were

used for each value. The increase in reliability found

closely approximates that predicted by the Spearman-Brown

formula if dual observations are used. This lack of effect

for interpolation is interpreted to be a result of the fact

that there was no trials effect in the data. As such MSE

for trials was very small and accounted for very little of

the total variance. The magnitude of the effect of the

interpolation on the trials variance can be seen by









comparing the trials variance in Step 2 and Step 3. Inter-

polation increased trials variance by more than 2 1/2 times.

In a case where time course across the night was important,

such an increase in resolution could be of extreme impor-

tance. In the present data interpolation by i-tself added

only about .03 to the reliability estimate.

The final control step was to eliminate effects from

the first night in the laboratory. The step added about

.06 to the single trial reliability.

The examination of the adjustment process, then, has

left the conclusion that removal of the waking threshold

and elimination of first night effects has added real

increases in reliability and that the smoothing effects of

interpolation increased reliability primarily through the

"artifact" of increased observation. If the Spearman-Brown

formula is used to correct the final Step 4 reliability

figure to that of a true single observation, that value is

.75. It is proposed that the base reliability of .61 was

improved .04 (to .65) by inclusion of waking threshold, .06

by exclusion of night 1 (to .71) and .04 (to .75) by the

interpolation. This figure would then predict a measure-

ment reliability of .95 for an average night of six trials

with the given adjustments.

Visual representations of depth of sleep results can

be seen in Figures I and 2. Figure 1 presents threshold

data as a function of time of night (Step 4) for two

subjects--the lightest and deepest sleepers of the six


































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subjects for their final three baseline nights. Each data

point represents arousal threshold minus waking threshold

at that point in time. The marks on the horizontal axis

represent the arbitrary points at which the night was sec-

tioned. Figure 2 is a plot for all six subjects of their

three-night average of data as taken from the time points

represented for two subjects in Figure 1. It can be inter-

preted as the average depth of sleep curve for the subjects

across their night of Stage 2 sleep. Measurement relia-

bility in Figure 1 is evidenced by the closeness of obser-

vations across nights within the subjects and the spread

between subjects.

In comparing the waking threshold data to the sleep

data (Tables 1 and 2), it can be seen that the increased

reliability in the waking data is more a function of

decreased within-subject difference than that of the also

decreased between-subject differences. There are two

possible reasons for this difference. It is possible that

"attention" is simply more variable in sleeping than in

waking subjects. It is also possible that the experimental

situation played a role. Arousals from sleep were distrib-

uted rather randomly across the night in what might be

described as a vigilance experiment with about eight

"signals." However, the waking threshold determinations

were time locked--they occurred immediately after arousal.

The night without sleep was designed to attack this point.

Data from the awake night, which actually lasted from









11:30 p.m. to 4:30 a.m., were transformed in a Step 3

analysis. An initial ascending value (waking) and a stable

waking value resulted. The'variance for each of these

measures was calculated and compared to the variance of the

arousal threshold and waking value recorded on the fourth

night of sleep during the same time period. The variance

of the arousal from sleep measures was greater than the

variance in both stable waking measures in all subjects,

and that difference was significant with an F-test for

variances (F.0,, > 6.39) in five of the six subjects.

The variance of arousal from sleep was greater in five

subjects than the variance for the initial ascending series

on the awake night, and that difference was significant

(F.0,, > 6.39) in four of the subjects. As no other

variance comparisons were significant, it must be concluded

that some process attributable to sleep tends to increase

not only absolute threshold but also the variability of the

measure when compared to waking values.

Several additional types of data were collected at

each arousal in the belief that they might relate to the

threshold. Those variables were body temperature, amount

of delta in the record, the length of the awakening and the

latency to sleep onset after the awakening. Within-subject

correlations for each item with the arousal threshold value

were done for random subjects. Patterns of significant

correlations were not found except in isolated subjects

with the exception of the relation between arousal





39


threshold and the time it took to fall asleep again after

the threshold procedure. These latency data are examined

in Appendix A.





EXPERIMENT 2


Hypotheses


Given a reliable measurement procedure (Experiment 1)

it was hypothesized that arousal in terms of both a motor

response (button push) and vocalization would be modified

after a single drug administration in Stage 2 sleep as

follows:

1) Flurazepam (30 mg) and pentobarbital (100 mg) would

elevate response thresholds over placebo depending

upon dosage and length of action.

2) Caffeine (400 mg) would lower response thresholds

depending upon dosage and length of action.

3) The sleeping medications were given in a standard

therapeutic dose; attempts to equate dosage on any

other dimension were not made; and any dose

differences reflect only that point. It was

hypothesized, however,that time course of activity

would be the major variable in identifying the

different compounds. Flurazepam was hypothesized

to have a short onset to peak activity from studies

of its time course effects on average frequency EEG

patterns. Caffeine, a short-acting stimulant given

at the equivalent of four cups of coffee, was also









hypothesized to show a short initial threshold

shift with a subsequent return to baseline values.

Pentobarbital, classed as acting for three to six

hours, might show a slightly later peak in activity

than flurazepam.

4) Pentobarbital, a barbiturate, and flurazepam, a

benzodiazepine, were chosen as a test of the model

developed by Bonnet (Note 1) and based on earlier

work by Routtenberg (1966, 1968). Pentobarbital,

working at the level of the reticular formation,

was hypothesized to decrease EEG responsivity up to

the point of waking threshold. Flurazepam, on the

other hand, was hypothesized to have less effect on

the ongoing EEG with the result that EEG desyn-

chronization would not be as rapidly followed by a

response on flurazepam nights as on pentobarbital

nights. These EEG/behavior disassociations would

also be a function of drug time course.


Method


The methodology of Experiment 2 completely paralleled

that of Experiment I with the exceptions to be noted here.

Experiment 2 was an eight-night design with six sub-

jects. Five of the subjects had participated in the first

experiment. The sixth subject had two adaptation nights

preceding this eight-night sequence. Experiment 2 took





place two months after Experiment 1. All laboratory condi-

tions were similar.

Of the eight nights, the first night was designated as

laboratory readaptation. Methods were exactly the same as

Experiment 1. Lab nights were three to five nights apart

(subjects coming on Monday night returned on Friday night).

On the last seven nights subjects received a pill at

11:15 p.m. when already in bed. Caffeine was given on

either night two or night eight and was only single blind.

On the other six nights subjects received a numbered, uni-

form pink capsule under double-blind conditions. The order

of administration was randomized within-subject such that

each subject had flurazepam, pentobarbital, and placebo in

one random order on the first three nights and in another

random order on the second three nights.

The same subjective report measures as used in Experi-

ment I were used with the exception that in Experiment 2

the Nowlis Mood Scale and the Spielberger State Anxiety

Scale were completed both in the evening and in the morning.

Three threshold measures were chosen to be examined.

The first corresponded to the Step 4 measure reported in

Experiment 1. It was the point of consistent correct

button push responses on an initial ascending series minus

a following measure of ascending threshold when awake

interpolated at six equally spaced points across the night

(12:39, 1:48, 2:57, 4:06, 5:15, and 6:24 a.m.). The

measure was planned to be a measure of possible shift in









sleep depth independent of any shift seen in waking

threshold due to conditions. The second measure was the

stabilized waking threshold'also reported in Experiment 1.

The third measure was designed to be a measure of "total

effect" on behavioral response. This threshold was calcu-

lated by using the stabilized ascending waking threshold

found at 11:15 p.m. each night. The 11:15 value was sub-

tracted from the intensity at which each subject verbally

said he was awake on all trials during the night. Thus

waking and sleeping effects attributable to drugs were

added in this measure, which is called the combined measure.

The code of drug conditions was devised by the

Hoffman La Roche Company and kept in the University

Infirmary in case of adverse reaction. It was returned

unopened to the Hoffman La Roche Company at the end of the

experiment. Upon receipt of the data reported here,

Hoffman La Roche supplied the conditions code.


Results


An analysis of variance was done on the data from each

of the three threshold measures. Effects for drug condition

(DRUG), trial across the night (TR), replication of the

experiment (R), subject (S), and all possible interactions

were found. Because only the placebo, flurazepam, and

pentobarbital were replicated, only those three conditions

were included in the initial ANOVA to keep all cells of the

analysis filled. The ANOVA's may be seen in Tables 3, 4,









and 5. Two striking results were found (unless otherwise

specified all tests and confidence intervals were chosen

at the .05 level of error probability). First, in terms

of pure sleep depth, waking threshold, and the combination,

there were significant main effects for drug condition.

Both drugs resulted in higher thresholds than placebo with

the exception of the effect of pentobarbital on waking

threshold, which missed being significantly different from

placebo by .14 dB. Second, in both measures involving

thresholds during sleep, there was an unexpected trials by

replications interaction. As no experimental conditions

varied from the first to the second half of the study and

a subject-by-subject examination of the data revealed no

obvious explanation for a replications effect, such an

effect was taken to represent a complex carryover effect.

Because effects of carryover or repeated use were not the

major topic of the present study and because significant

drug condition effects did exist, it was decided that a

further examination of the first administration of each

drug was most proper.

Tables 6, 7, and 8 report the first administration

analyses of variance for placebo, caffeine, flurazepam, and

pentobarbital. A trial-by-trial plot of the three types of

threshold drug may be seen in Figures 3, 4, and 5. In the

waking condition, again, main effects for drug condition

were found. The waking threshold after caffeine (7.58 dB)

was less than that after placebo (10.17 dB), and the





Table 3. Replication by Drug Condition by Trial by Subject
ANOVA for Flurazepam, Pentobarbital and Placebo
from "Pure" Sleep Data


Source



SxR
DRUG

SxxDRUG

TR
SxTR
RxTR
SxRxTR
DRUGxTR
SxDRUGxTR
RxDRUGxTR
SxRxDRUGxTR

TOTAL


10164
16
494
1318
214
440
542
764
140
403
89
77
98
151
136

418


Significance
NS


RxDRUGxTR
3-wy iterctin 10,50 SxRxDRUGxTR


2) 2-way interaction F 11


.812


- 4.540


.783


= 6.170


NS


.005


NS


RxDRUG
SxRxDRUG


RxTR
10,25 SxRxTR

FDRUGxTR
10,50 SxDRUGxTR

DRUG
F2,10 Sxyhy@


3) Main effect


Drug Condition Means


Placebo

Flurazepam

Pentobarbital


42.06 dB .05 Confidence Interval = 6.57 dB

50.10 #

48.63 # # Greater than placebo using
t confidence intervals





Table 4. Replication by Drug Condition by Trial by Subject
ANOVA for Flurazepam, Pentobarbital, and Placebo
from Waking Threshold Data


Source



SxR
DRUG
SxDRUG
RxDRUG
SxRxDRUG
TR
SxTR
RxTR
SxRxTR
DRUGxTR
SxDRUGxTR
RxDRUGxTR
SxRxDRUGxTR

TOTAL


MS

2430
36
79
221
43
9
31
20
8
4
6
11
10
12
11

72


Significance


RxDRUGxTR
1) 3-way interaction F = 1.04
10,50 SxRxDRUGxTR


=RxDRUG
SxRxDRUG


2) 2-way interaction F
2,10


=.29


=.72 NS


=1.08 NS


.45 NS


=5.18 .03


=2.46 NS


RxTR
5,25 SxRxTR

F = DRUGXTR
10,50 SxDRUGxTR


71,5 SxR

DRUG
2,10 SxDRUG

TR
F5,25 STR


3) Main effect


Drug Condition

Placebo
Flurazepam
Pentobarbital


Means


10.95 dB
14.40 #
13.24


.05 Confidence Interval = 2.43 dB

# Greater than placebo (.05)
using a t confidence interval





Table 5. Replication by Drug Condition by Trial by Subject
ANJOVA for Flurazepam, Pentobarbital and Placebo
from Combined Waking and Sleeping Data


Source



SxR
DRUG
SxDRUG
RxDRUG
SxRxDRUG
TR
SxTR
RxTR
SxRxTR
DRUGxTR
SxDRUGxTR
RxDRUGxTR
SxRxDRUGxTR


17244
1005
543
3319
402
233
281
953
138
179
58
67
133
88
101


TOTAL


1) 3-way interaction F1,0


Significance
NS


RxDRUGxTR
SxRxDRUGxTR =


.872


2) 2-way interaction F2,0


.830


= 3.060


=.510


= 8.260


RxDRUG
SxRxDRUG


RxTR
5,25 SxRxTR

F=DRUGx~TR
10,50 SxDRUGxTR


F2,10 SxRUS


3) Main effect


Drug Condition

Placebo

Flurazepam

Pentabarbital


Means

46.50 dB .05 Confidence Interval = 7.44 dB


59.68 #


55.92 # # Greater than Placebo (.05)
using a t confidence interval

















ID
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r-

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in

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m
o

m
to


nAA
HNM







O


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m



oo

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W c-na moo ar
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lu Immlnmlnv m

















ax

U 3 x3 D 0


O eNx M xx O
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E -r m
0d a CO
a a, O
O a, la c -He
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to a
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Table 7. Drug Condition by Trial by
Flurazepam, Pentobarbital,
from Waking Threshold Data


Subject ANOVA for
Caffeine and Placebo



MS


Source


S U
DRUG
TR
SxTR
DRUGxTR
SxDRUGxTR

TOTAL


1374
320
19
13
11
9
7

64

Significance


=1.29


=17.2


DRUGxTR
SxDRUGxTR


1) 2-way interaction F
15,75


NS


.0001


2) Main effect


DRUG
F
3,15 SxDRUG


FS,25 STR


=1.15


Drug Condition

Placebo

Flurazepam

Pentobarbital

Caffeine


Means


10.17 dB


14.31

12.89


C

*.05 level difference using a t confidence interval
Confidence Interval = 2.20 dB





N m

V a VlmN L
v I v v


E N m \o Npl I






amo u m-r o
10 mo amm m
N mua







O~ mm mma



~a, ca



o 4r
-0m v



lcl O~ Nx~I O~ a




ZS~~~ a m- vr~d

a) m N N 0


100 e

ScE a

~~~1~ E0 OC-o
~O O 0 m v -He


U~~~ C1P PP F0 C
E~~0 0i O-H
art ~ ~ o eAp~ C ct






































'e--
C


F `\


SFlurazepor
.,- "Pentobarb
g.\ Placebo
Pe -1Caffeine


n - -
,ital ----


60-











450-


Confidence


'*-- -


1
(12 39)


(1:48)


(2:57)


(4:06)


(5:15)


6
(6:24)


TRIAL




Figure 3. Average Depth of "Pure" Sleep Across the Night
after Administration of Flurazepam, Pento-
barbital, Caffeine, or Placebo





Flurazepam -
Pentoborbital -----
Placebo
Ca ffe in e ----


,~-----~--,
~ c


PE


PL .--*


,*


I
(12:39)


2
(I 48)


34
(2:57) (4 OS)
TRIAL


5
(5:15)


6
(6:24)


Figure 4. Average Threshold of Awake Subjects Across the
Night after Administration of Flurazepam,
Pentobarbital, Caffeine, or Placebo









































TIME


I


801


Flurozeparn ---
Pentobarbital --
Placebo ---
Caf feine ---.


's Conl


Pe


_


c 70'

-I
co 60-


>



5-



30?


fidence
Iterval --+


a.
~.-~ g


...e........ ..


c


I
(12:39)


3
(2:57)


4
(4-06)


2
(I=48)


5 6
(5: 15) (6- 15)


Figure 5. Average Combined (Waking and Sleep) Threshold
Across the Night after Administration of
Flurazepam, Pentabarbital, Caffeine, or Placebo





threshold after placebo was less than that after either

pentobarbital (12.89 dB) or flurazepam (14.31 dB). There

were no trial effects.

There was a trial by drug condition interaction in

both of the sleep measures. Confidence intervals were

computed, and trial-by-trial comparisons were made both

across trials and across conditions. Those results are

noted in Tables 6, 7, and 8 at the foot of each column and

the end of each row. Briefly, condition differences in the

"pure" sleep measure were seen only for the first four

trials. In the first two trials administration of

flurazepam and pentobarbital resulted in higher thresholds

than the administration of caffeine or placebo, which did

not differ. Thresholds after caffeine administration

remained lower than those after the other two drugs in

Trial 3 but were lower than only pentobarbital by Trial 4.

Trial effects were seen only within flurazepam and pento-

barbital. An initial peak was seen in both which differ-

entiated the first trial from the last three or four trials

and the second trial from the last two or three trials.

The results for the combined threshold measure, as

would be expected, contain components found in both the

waking and "pure" sleep measures and are therefore more

extensive than either of the others. Thresholds after

caffeine were lower than those after flurazepam for all six

trials and less than placebo and pentobarbital for all but

Trial 5. Placebo thresholds were lower than those for









1urazepam for the first three trials and Trial 6 and were

lower than those for pentobarbital on the first two trials.

Of all the threshold comparisons, flurazepam and pento-

barbital differed significantly only on the first trial in

the total data, where thresholds were higher with fluraze-

pam. In the combined measure there were trial effects

(time of night) for all conditions including placebo.

Placebo was marked by one low threshold trial (Trial 6) and

caffeine by one high threshold trial (Trial 5). The picture

for flurazepam and pentobarbital was very similar to that

seen in the "pure" sleep data--higher thresholds on the

first two or three trials than on the last two (see Table 8

for the complete rundown).

To more fully address Hypothesis 3 (differentiating

drugs by time course effects), trend analyses (Note 4) were

done for the three threshold measures. The intent was to

see if additional differences in the form of drug activity

in trials across the night could be found. Illustration of

all results should be referred to Figures 3, 4, and 5,

which graphically display the various trends in the three

threshold measures. The trend analysis yields an F

statistic with 3 and 5 degrees of freedom if the strict

criteria of Box, which allow only n 1 ("n" being the

number of different subjects) degrees of freedom for error,

are followed. A more lax criterion, commonly used in

analysis of variance, is to allow (G 1)(n 1) degrees of

freedom for error (where "G" refers to the number of









groups) even if the same subjects appear in all groups.

Using the strict criterion an F of 5.41 is required for

significance (.05 level) in the present data, and only the

linear differences (slope) between both flurazepam and

pentobarbital as compared to the linear trend in the

caffeine condition in the "pure" sleep data (Figure 3) meet

this criterion (F = 10.39 and F = 7.39 respectively).

Using the less stringent criterion (F0531= 3.29),

flurazepam has both linear characteristics which differ

from the linear characteristics in the placebo and also

curvilinear characteristics which differ from those seen in

placebo. Specifically some quadratic curvilinearity can be

seen throughout the placebo condition and a reverse

quadratic is seen in the final flurazepam trials.

In the waking data (see Figure 4) all significant

trend effects involved the pentobarbital data. The linear

trend in pentobarbital differed from both caffeine and

flurazepam conditions. In addition the quadratic curve in

the placebo condition allowed it to be differentiated from

the reverse quadratic effect seen in the last five trials

of pentobarbital. Finally a quartic component (Cul

versus Lrk) ) was found between pentobarbital and

flurazepam indicating an early, middle, and late peak for

flurazepam (Trials 1, 3 and 6) versus middle peaks for

pentobarbital (Trial 2).

In the data combining sleep and waking effects

(Figure 5), the linear effects of both flurazepam and









pentobarbital differed from caffeine and the pentobarbital

also differed from placebo. Both flurazepam and pento-

barbital, by virtue of early peaks, differed from caffeine,

which had a late peak, in the cubic trend.

Tests of Hypothesis 4 were made by visual examination

of several events in the EEG records for evidence of

disassociations. Stimulus intensities were recorded at

the first point of breakup of EEG sleep patterns, at the

point of the beginning of button push responses and at the

point of verbalization. Alpha production was examined

around the button push and verbalization points as well.

Previous work has not indicated time course effects in any

of these variables, but because the drugs might institute

time course effects a simplified procedure to control for

such possible effects was deemed advisable. Each night was

split into five parts as a function of time, and all values

falling within that time period were averaged within each

subject within each condition. The groupings were as

follows: Group 1 5 80 minutes, Group 2 > 80 minutes and

c 180 minutes, Group 3 > 180 minutes and < 280 minutes,

Group 4 > 280 minutes and < 380 minutes, and Group 5 > 380

minutes. An initial subjects by replications by drug

conditions by group ANOVA was done. The only significant

effects were found in the measure of the difference between

initial breakup of EEG patterns and the point at which

subjects verbally said they were awake. Because all inter-

action terms containing replications and the replications






59


main effect were nonsignificant, the data for the two

replications on flurazepam, pentobarbital and placebo were

combined within condition so that they could be compared

with the caffeine condition in an analysis with minimal

empty cells. The results of this subject by drug condition

by group ANOVA can be seen in Table 9. An increasing

tendency for initial body movement to be disassociated from

reported awakening can be seen as one moves from caffeine

to placebo to flurazepam to pentobarbital. The difference

between caffeine and the other drugs is significant, and

the difference between placebo and pentobarbital just

misses significance.






60


Table 9. Drug Condition by Trial by Subject ANOVA for the
Disassociation of EEG Breakup and Verbalization
as a Function of Tone Intensity


Source df MS

S 5 1395
DRUG ;3 842
SxDRUG 15 195
GROUP 4 251
SxGROUP 20 63
DRUGxGROUP 12 88
SxDRUGxGROUP 57 49

TOTAL 116

Significance

1) Interaction F = R~GOP = 1.77 NS
12,57 SxDRUGxGROUP

2) Main Effects F DRG= 4.32 .0
3,15 SxDRUG

F GOP=4.01 .02
4,20 SxGROUP

Drug Condition Means

Placebo 8.72 dB
Flurazepam 14.56*
Pentobarbital 16.21*
Caffeine 4.40*

*Caffeine differs at .05 level


Group (Time across the Night) Means

1 6.24** dB
2 13.35**
3 14.51**
4 11.69**
5 9.90


**Group 1 differs at .05 level















DISCUSSION


The two major purposes of the present work were first

to estimate the reliability of a threshold measurement of

the sleep process and second to examine the effects of

common drugs on threshold. The reliability of the measure-

ment process could only be classed as little short of

remarkable given the single, small subject class. With

appropriate controls the data indicate that from a single

night of measurement (six trials) in a laboratory-adapted

subject measurement reliability is estimated to be

r6 = .95.
In one final reliability analysis using data (Step 4

transformation) from the five subjects participating in

both experiments, an average threshold value was found for

each subject from the last two baseline nights in Experi-

ment I and from the two placebo nights of Experiment 2.

The Pearson r was .804. The figure implied a degree of

reliability in a two-night sample over a two-month period,

but a reliability estimate for a single trial would have

been lower than for closer nights.

There is also little question that flurazepam, pento-

barbital and caffeine affected responsiveness during sleep

when compared with placebo in the present experiment.









Further, the ability to make a statistical statement with

six subjects is indicative of a gross effect as can be seen

by the size of the confidence intervals employed in the

sleep threshold data (approximately 12 dB). The more

telling and perhaps more important findings most probably

lie in two other interrelated dimensions. Those dimensions

are the interplay of drug effects on "pure" sleep as

opposed to waking thresholds and the time course effect of

drug action.

The results to be discussed with noted exceptions are

those which achieved a degree of statistical significance.

In a study with a very small number of subjects, statistical

significance is often indicative of either a very gross

effect or chance. In a single study it is impossible, of

course, to certainly separate the two. In a later part of

this section the attempt to replicate the present results

will be discussed, and from both sets of data, the overall

effect for drugs appears to be a gross effect. However,

the individual time course effects and trends are succeed-

ingly less likely to be found to be gross effects and are

more likely to be idiosyncratic to this experiment. A part

of this effect comes from the unquestionably inflated alpha

intervals from the multiple comparisons described here.

Such an avenue was chosen because it was considered more

important to trace possible variables which may be borne

out by replication than to ignore such variables and risk

a similar fate for them in future replications. The









present discussion, then, is certainly descriptive and

hopefully predictive for replications in several important
areas.

As judged from significance levels in the ANOVA

tables, the major effect of caffeine (as compared to

placebo) is on waking threshold. When awakened after

caffeine administration subjects had lower thresholds

than when awakened in any other condition, but the

"pure" sleep measure did not differ from placebo. The

present data does not allow speculation as to the subjects

being "more awake" in the caffeine awakenings. It

should be stressed that the same criterion of a stabilized

threshold and waking EEG was always used but that the

EEG criteria were difficult to control because a subject

with his eyes open and/or concentrating on a threshold

task may produce little alpha although awake. Also,

in some conditions subjects had a difficult time staying

awake for the three seconds between tones. The final

question is a difficult one never completely resolved

at a practical level--was the subject really awake?

Obviously, spindle and K-complex activity was never

included in a "waking" threshold. But latencies to

spindles were in some conditions incredibly fast (see

Appendix A); threshold could jump 70 dB in three seconds,

and the data on waking thresholds must be viewed in that

realistic context. Let it suffice to say that it occasion-

ally took several awakenings to get subjects to both wake









up and stay awake for the few requisite seconds that form

the basis for what is called waking threshold.

The more fine-grained trend analyses found the

slightly positive linear slope of the caffeine nights to

be different from the larger negative linear slope of the

other drugs in the sleep data. Both effects, of course,

were expected as indicators of lessening drug effects over

time. Still the trend analyses could not differentiate

caffeine from placebo.

Flurazepam and pentobarbital present a picture quite

different from that of caffeine. Because the analyses of

variance could separate these two drugs at only one point

(higher thresholds for flurazepam in the first trial of the

combined data), their ANOVA effects can be explained

together. Both were characterized by high peaks at the

first trial point (1 hour and 24 minutes after drug inges-

tion) in the pure sleep and combined data, and those peaks

remained significantly higher than placebo for the first

two or the first three trials respectively. In addition,

both drugs resulted in higher waking thresholds than did

placebo.

These drugs additionally displayed clear time of night

effects in both sets of sleep data. Thresholds were always

higher on the first two or three trials than they were on

the last two or three trials. This may be contrasted with

placebo and caffeine conditions, which both had only one









extreme point that may have occurred by chance. The

placebo condition will be discussed further in a later

section.

The trend analysis picture of flurazepam versus pento-

barbital is an intriguing one with implications for differ-

ential effects on basic sensory processing and with ties to

other data bases. An unstated hypothesis apparent in the

present treatment of results is that waking threshold and

"pure" depth of sleep are to some extent independent

measures. There is, for example, an approximate zero

correlation between the two measures in baseline data. To

the extent that an independence exists, the measures could

be selectively influenced by many variables. In the

present case all drugs modified waking thresholds. The

effects of caffeine and flurazepam were fairly linear across

the eight hours of sleep, but the effects of pentobarbital

(as gleaned from trend analysis) began to dissipate about

the fourth trial. This trial was about five hours after

drug ingestion, and the predicted duration of pentobarbital

action (from the pentobarbital package insert) is 3-6

hours. The trend analyses also uncovered a quartic effect

between flurazepam and pentobarbital in the waking data.

If quartic curves are examined, it is seen that the maximum

difference is found in a set of curves which display both a

steep onset and a steep ending in opposite directions. In

this case flurazepam has been fit with a curve Lr\ and

pentobarbital with a curve (11 In terms of drug time









course, this says that flurazepam has a faster onset than

pentobarbital (as is borne out by the significant difference

in those two drugs in the first trial in the combined data)

and continues to act for a longer time. Both of these

results are borne out by aspects of sleep latency (Appendix

A) and questionnaire data (Appendix B).

A few studies have examined performance on a battery

of tasks in the morning 8-10 hours after drug administra-

tion. In two studies, Bixler, Leo, Mitsky, Pollini, and

Kales (1974 abstract) first reported no performance effects

(tasks not specified) in the morning eight hours after

ingestion of either flurazepam (30 mg) or secobarbital

(100 mg). In a later study with about twice as many

subjects, Bixler, Leo, Mitsky and Kales (1976 abstract)

found performance decrements with flurazepam (30 mg),

secobarbital (100 mg) and phenobarbital (100 mg) as com-

pared to placebo. The different results were explained

as a result of allowing subjects to eat breakfast before

testing in the first study. Roth, Kramer, and Lutz (Note 7)

reported the effects of flurazepam, triazolam, secobarbital

and placebo on performance. Ten hours after drug admin-

istration performance on a battery of tests including

arithmetic, digit symbol substitution and card sorting was

significantly impaired by only flurazepam (30 mg) as com-

pared to placebo. The weight of evidence from these studies

directly supports the present evidence for continued action

of flurazepam until the end of the sleep period (and









longer) and offers minimal support that the barbiturate

effects may be shorter lasting. Further, the present

results indicate that the reported performance decrements

are a direct result of sensory mediated deficits.

In the sets of sleep data, no trend analysis was able

to split the effects of flurazepam and pentobarbital.

However, in the pure sleep data, a late quadratic effect

was seen with flurazepam as compared to placebo. This

levelling effect, most apparent in the last three trials,

seems to indicate a levelling effect of flurazepam on

sleep. However, this result is confounded by the fact that

the final trial threshold with flurazepam is almost 10 dB

higher (nonsignificant) than the last placebo observation.

If the trend find is appropriate, it would indicate differ-

ential effects of flurazepam on sleep and waking thresh-

olds. The waking effect continues through Trial 6 while

the sleep effect has dissipated by Trial 4. Regardless, a

strong trials effect was seen in the sleep data and vir-

tually no trials effect was seen in the waking data. In

the pentobarbital, on the other hand, the trials effect on

the pure sleep data does appear to be (nonsignificantly

from ANOVA) reflected by a downward shift in waking

threshold on Trial 4. Because waking thresholds have not

been previously examined with rigor in sleep studies, the

present evidence for independence in sleep and waking

measures is the only evidence either for or against such a

contention. A final answer must rest in replication. On









the basis of the less ambiguous waking results, the present

conclusion would be that flurazepam is both faster acting

and longer lasting than pentobarbital.

Several previous studies have reported a time of night

effect for sleep depth in both REM and Stage 2 sleep under

baseline conditions. Most of those studies have dichoto-

mized data (early/late) in their comparison and found

thresholds from the early part of the night to be higher

than those from the later part. In data from Experiment I

and the baseline and placebo nights in Experiment 2, a

quadratic effect was always seen in the data, although with

one exception a trials effect was never present in the

analyses of variance. If the Experiment 1 data are dichoto-

mized and the values of each subject are averaged for the

first half and the last half of the four baseline nights,

the group means show thresholds higher in the first half of

the night in the combined data (51.1 dB versus 48.4 dB) and

in the pure sleep data (36.1 dB versus 32.2 dB) but not in

the waking data (15.0 versus 16.2 dB). None of these

differences was significant, and none was significant even

if the first trial of the night was excluded from the

"early" data. The differences reported in the literature

have been small, and the lack of significance here could be

a result of the small sample or in the selection of awaken-

ing times. Thirdly, a methodological question could be

raised. Evidence dating from the 1930's (Mullin et al.,

1933; Mullin et al., 1937) has shown that depth of sleep








varies for up to 25 minutes after an arousal and also that

body movements increase strikingly across the night. If

sleep depth also varied after body movements and studies

were not controlled for signal presentations shortly after

body movements (a 10-minute criterion was set in the present

study), more extreme low values might be found in the

second half of the night corresponding to an increase in

the probability of a body movement.

One of the reasons for the choice of flurazepam and

pentobarbital in the present study was to examine the

effects of two classes of drugs on EEG arousal character-

istics. While there was some tendency for both drugs to

disassociate the verbal response from the first breakup in

the EEG as compared to placebo and more certainly caffeine,

the two drugs could not be differentiated by any EEG

arousal characteristics. There are two possible reasons.

Most simply it could be that differential effects on behav-

ior just do not exist although this does not agree with

evidence from Kornetsky and Bain (1965). An equally possi-

ble and more plausible explanation is that the data analysis

procedure, which consisted primarily of visual alpha counts

before and after button push and verbal responses, was

grossly insensitive. This last possibility could be tested

in future studies by the use of computerized frequency

analysis, but the meaningfulness of such an analysis would

be limited because it is already known that these two drugs

differentially affect some aspects of the EEG. Any finding









on differing arousal characteristics would be confounded by

that knowledge.

The failure to support'the dual arousal system

hypothesis coupled with the present and other evidence of

within-subjects shifts in depth of sleep under highly

controlled EEG specifications leaves the present results

in a theoretical quandary. The evidence showing EEG/behav-

ior disassociation is extensive. The present results

indicate that that disassociation cannot be easily iden-

tified in terms of EEG arousal characteristics except for

the EEG breakup/verbal response dichotomy. Both the

present results and the Kornetsky and Bain finding could

be reconciled in a framework which posited that information

from both (or all) arousal systems contribute EEG patterns

in an averaged fashion so that inhibitory effects in either

of two systems could have a similar EEG effect. But these

rules might not apply to actual behavior as tested by

Kornetsky and Bain (1965). A test of this hypothesis would

require a more clearly-defined signal detection task in

addition to the awakening threshold collected in the

present study. As a rough estimator, there was a trend for

more button push responses to be made before a verbaliza-

tion in the present study after pentobarbital than after

flurazepam (means 3.08 versus 2.04 responses, t = 1.66, NS)

in agreement with the similar findings of Kornetsky and

Bain. Perhaps the most important conclusion, however, must

be made a step further away from the data. The present









results indicate an EEG/behavior discrepancy after drug

use. This is exactly predicted by a large amount of drug

work dating to 1938 which has shown behavior/EEG disas-

sociation after drug use. The importance of the many

EEG/behavior disassociations is not the mechanisms by which

they are caused but rather their mere existence and pro-

clivity. Their existence raises doubts about the validity

of EEG as a measure under important conditions. The EEG

studies of flurazepam and the benzodiazepines in general,

for example, have indicated a tendency for suppression of

slow wave sleep and little effect on REM. These results,

indeed, were found in a study by Itil et al. (1974). In

that study several doses of benzodiazepines and placebo

were given and a significant but seemingly paradoxical

positive correlation was found between subjective "light-

ness" of sleep and the amount of Stage 4. This correlation

is understandable if the possibility that the benzodiaze-

pines both decrease Stage 4 and increase behavioral thresh-

old irrespective of sleep stage is considered. In short it

is possible to increase subjective "depth" of sleep and

behavioral threshold while decreasing Stage 4, which nor-

mally has been associated with deep sleep in humans. The

implication is that EEG as a single measure of sleep may be

deceptive at best and inappropriate at worst when it is

confounded by the experimentally-produced artifact of drug

use or other trauma.





The present picture of drug activity differs consider-

ably from standard EEG results. That difference is a

difference of focus and methodology and, of course, is a

difference of question asked.

Experiment 2 was designed to have a replication of

flurazepam, pentobarbital and placebo effects within

itself. This replication failed on several dimensions.

In the initial analyses of variance, two significant

effects were found. One, for drug conditions, has been

discussed at length. The other, a trials by replications

interaction, has not been examined. A replications inter-

action means that somehow the first half of the experiment

was different from the second half. When confidence inter-

vals were constructed in the "pure" sleep threshold data,

it was found that thresholds on the first trial of the

second replication were lower than thresholds on the first

trial of the first replication. A similar finding, extend-

ing for three trials, was found in the combined data. Both

EEG and subjective report data bases uphold the contention

that the second replication was different from the first.

In terms of EEG effects, initial latencies to Stage I sleep

onset were ranked within each subject for the eight experi-

mental nights. When the ranks were compared, it was found

in both the placebo and pentobarbital conditions that the

rank latency for all subjects was greater in the second

replication than in the first replication (there was one

rank tie for placebo, but the sign test for n = 5 was still









significant), an effect significant at the .02 level in a

sign test. There was only one reversal in the flurazepam

group, but that made the results nonsignificant. On the

Post-sleep Inventory, 6 of the 30 items differed from

replication one to replication two. Those items included

reports that subjects had more thoughts at the time of

going to bed and during the night, thought the room temper-

ature and the bed were less comfortable, and thought that

they tossed and turned more in replication two.

There are several possible reasons for a failure of

replication in the present study. Five will be briefly

discussed here although it is certainly possible to find

others and no clear answer will emerge. Perhaps the most

well-founded explanation is a tolerance explanation. While

previous studies have used a similar three-night minimum

washout between drug conditions (Itil et al., 1972, 1974)

and a three-night examination of recovery effects from

extended use is a standard Kales' procedure, it is possible

that effects continue longer than Kales has measured and

that the Itil studies could not or did not examine toler-

ance effects. A tolerance explanation would predict longer

latencies on the second drug night and is considered a

conceivable explanation to Roth (Note 6) from work he has

done with flurazepam. Another explanation is a "time of

the quarter" explanation. All subjects were students and

might have been under increasing academic pressure as the

quarter progressed. The final night on which flurazepam,









pentobarbital or placebo was given was on the Monday of

dead week. Such an explanation would posit subjects had

more thoughts and longer latencies because they were more

worried about school in the second replication. Obviously

it cannot be shown if more thoughts caused longer latencies

or longer latencies caused more thoughts. A third explana-

tion is an "end of experiment" explanation. Subjects spent

13 nights in the lab over a three-month period. For some

subjects the last three nights were the second drug repli-

cation. As such, their dissatisfaction with nonoptimal

laboratory accommodations (uncomfortable bed and tempera-

ture) could have increased sufficiently to interfere with

their sleep process. This explanation is probably doubtful

because adaptation trends usually go the other way and do

not reverse. Roth (Note 6) has commented that end of

experiment effects have occasionally been encountered in

his work but that they are usually seen after multiple con-

secutive nights, which did not occur in the present study.

Fourthly, the threshold data could be explained by state

dependent learning within drug states. However, this argu-

ment would not predict a latency or a subjective sleep

change and could probably be discounted for those reasons.

Finally, the early trial threshold shifts could be explained

by the increased sleep latencies. In a time sense, peak

threshold obviously depends upon latency and the data was

not corrected for that factor. The thresholds of the

second replication, which came, on the average, more





closely to sleep onset, could be lower for that reason, but

this explanation fails in that it cannot explain why the

latencies were longer.

In short, no single, satisfactory explanation of

replication effects has presented itself. An examination

of individual data is no more enlightening. However, this

discussion has brought forward, as an assumption, a very

important fact. Three relatively independent measures of

the sleep process have been discussed. It is indeed very

relevant that when an effect (hypothesized or not) was

found in the threshold measure, it was directly supported

by findings in both EEG and subjective measures. Such a

finding not only documents the existence of an underlying

factor, but also validates the measures used.
















S UMMIARY


The present study has demonstrated high reliability

in a threshold measure of the sleep process. It has

further documented the effects of flurazepam and pento-

barbital in increasing auditory thresholds of sleeping and

waking subjects in a time course fashion as compared to

placebo. Effects of caffeine in decreasing auditory

thresholds were also seen. Trend analyses indicated that

flurazepam was both a faster and a longer-acting drug than

pentobarbital but those results (and really all results in

a six-subject study) must be considered tentative.

Flurazepam and pentobarbital could not be separated by

their EEG arousal characteristics. The finding was con-

sidered in opposition to a dual-arousal system hypothesis.

Finally, failure to achieve a complete replication of all

time course effects was taken to represent possible

tolerance or time of the academic quarter effects and

also as validating evidence of three separate measures of

the sleep process--EEG, subjective report, and behavioral

threshold.





APPENDIX A

THE EFFECT OF FLURAZEPAM, PENTOBARBITAL
AND CAFFEINE ON LATENCY TO SLEEP ONSET
ACROSS THE NIGHT


The latency to sleep onset at the beginning of a sleep

period is reported in virtually every EEG study of sleep.

An estimate of how much time subjects spend awake over a

period of sleep (Stage 0) is also routinely reported. Both

types of data are also routinely collected on almost all

sleep questionnaires.

Students of insomnia have used both latency and stage 0

time as criteria for assessing the degree of an insomnia

problem. They have also used a third measure, usually

-referred to as early awakening and inability to fall asleep

again. While at least one source is available (Webb &

Agnew, 1975) that shows a circadian effect on initial

latency to sleep onset, a systematic study on the speed

with which subjects fall asleep after an awakening during

the night has not been done. It was proposed that such

sleep latency measures across the night might be an impor-

tant variable in determining drug time course and effec-

tiveness.





Method


As a part of the earlier reported experiments,

subjects were awakened from Stage 2 sleep several times

during the night. When each subject was awake, the

experimenter turned on a small night light in the subject

room and measured the subject's waking threshold. Subjects

had been instructed that their waking threshold would be

tested at each awakening, and that they were to remain

awake and work on the threshold task as long as the night

light was on. The turning off of the night light was the

signal that the threshold determination was complete and

that the subject should fall asleep again. Length of

awakening (i.e., length of threshold determination) was not

controlled. The criterion for awakening was rather a

stabilized waking threshold where stabilized was defined as

the same or close (5 dB maximum) value on three successive

ascending series in the threshold determination. With rare

exceptions this criterion was met.

As sleep stage of awakening was controlled, the

absolute time of night of awakening could not be controlled.

Because Webb and Agnew (1975) had displayed circadian

effects in sleep onset, each night for each subject was

arbitrarily split into five parts by time (0-80 minutes,

81-180 minutes, 181-280 minutes, 281-380 minutes, 381+

minutes after an 11:30 p.m. bedtime), and all observations

for each subject for each condition were averaged within





each time block to get a single subject value for each time

block for each condition. The few empty cells were filled

with the average of blocks on each side. An empty cell in

the first or last time block was left unfilled.

Latency was measured as the time from when the night

light was turned off to the appearance of the first spindle

or K-complex in the record. Latency values are invariably

skewed. As a result all within sleep latency values for

each subject were rank ordered across all conditions, and

it is those ranks which will be reported as data.


Results


As in previous data analyses an initial ANOVA was run

primarily to test for a replications effect in the

flurazepam, pentobarbital and placebo data. No significant

interactions were found, but there were significant main

effects both for drug condition and time of night. Because

there was no replications effect, data for the placebo

nights and corresponding drug nights were averaged and

entered into a second analysis of variance also containing

data from the single caffeine night. That analysis is

presented in Table 10. Three things should be noted about

the ANOVA. First, the mean square for subjects does not

equal zero as it should for ranks because of averaging over

conditions, because the laboratory adaption night was

ranked but not entered into the analysis and because cells

were averages of ranks. Second, there were eight empty


















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cells. Third, there was a significant drug condition by

time of night interaction (F = 2.90). Individual time by

condition comparisons were made. The latency rank by time

plot can be seen in Figure 6 for the four drug conditions

and differences are noted in Table 10. Briefly, latencies

were very short with flurazepam throughout the night with

a relatively small time-of-night effect.

At the second and third time points placebo, fluraze-

pam, and pentobarbital did not differ, but with flurazepam

latencies remained short throughout the sleep period.

Pentobarbital latencies differed from placebo only in the

first time period. Latencies after caffeine administration

were longer than all other conditions for the first three

trials and longer than flurazepam for all trials. Definite

time of night effects were seen in all conditions except

flurazepam. Latencies in the early and late parts of the

night were high, and with placebo and pentobarbital

latencies in the second and third periods were lowest.

Initial sleep onset latency has been briefly dis-

cussed. It was not included with latency during sleep

results because stimulus conditions differed, values were

generally higher, and there was a replications effect in

that variable. The median rank for initial sleep onset

latency corresponded to an average latency of about 6.7

minutes. During the sleep period a similar median rank was

about 1.5 minutes. Only 6 percent of the sleep period

latencies were longer than 6.7 minutes, which indicated





83






4 5 *-
C =.Flurazepom ---
~~Pentobarbital --
Placebo
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little overlap of distributions. However, there were

condition effects in the initial sleep latency data. In a

night-to-night comparison a sign test requires no reversals

for a statistical level of significance to be reached with

six subjects. Latencies to initial sleep onset were

significantly longer after caffeine than after flurazepam

or after the first pentobarbital administration. There was

one reversal in the comparison of caffeine to the first

placebo night (caffeine night longer) and one reversal in

comparison of the first flurazepam night to the first

placebo night (placebo night longer).


Discussion


Circadian and drug effects on sleep onset are obvious

and profound. The time of night differences may be

explained to be a dual function of circadian and sleep

process effects. For this reason, the present results are

not directly comparable to the Webb and Agnew (1975) data,

which examined only circadian effects on initial latency.

The Webb and Agnew data suggested a circadian trough in

sleep latency somewhere around 7 a.m. The present data

suggest an earlier trough (around 1:30 a.m.), which may be

the result of the interaction of circadian and sleep process

effects--i.e., it is very hard to fall asleep after just~

having slept for 10 hours. Long latencies in early and

late parts of the night would then reflect initiation and

termination of the sleep process effects and the curvi-

linearity of the latency function would correspond to the








curvilinearity in threshold. This would underscore the

biorhythmic aspects of the sleep process without necessarily

supporting any theoretical position. Further work is

obviously needed to examine sleep process effects on

latency in sleep placed at different circadian times to

test the relative contributions of both factors.

The relatively small circadian influence seen after

flurazepam administration and the lack of condition differ-

ences (between placebo, flurazepam, and pentobarbital) at

the second and third time points is probably indicative of

a basement effect. The average latency in the six subjects

for a rank of 15 is slightly under a minute. Since latency

was measured as the time to the production of a spindle or

K-complex and spindles or K-complexes may occur only two or

three times during a minute of normal Stage 2 sleep, an

average shorter than about 30 seconds is probably impossible.

Latencies are approaching this value in the 12:45 to

2:30 a.m. range of the data except after caffeine adminis-

tration.

The latency data, as presented in Figure 6, offer

interesting corroborative information concerning drug time

course of action. In the combined threshold data (sleep

plus waking thresholds) of Experiment 2, caffeine differed

from flurazepam on every trial just as caffeine differs

from flurazepam on every trial in the latency data.

Caffeine differs from placebo in the combined threshold

data for the first four trials only (until 4:06 a.m.).

In the latency data caffeine differs from placebo for the





first three trials (until 4:10 a.m.). In the threshold

data, flurazepam differed from placebo on the first three

trials and the last trial. 'In the latency data it differed

from placebo on the first trial and on the last two. Only

the comparison of flurazepam with pentobarbital does not

follow the earlier script exactly. From the conclusions of

Experiment 2 it would be predicted that flurazepam would

differ from pentobarbital in that latencies after its use

would be shorter than after pentobarbital in the first and

last time period because flurazepam was predicted to have

faster initial activity and to last longer than pento-

barbital. As predicted, latencies after flurazepam were

shorter than after pentobarbital at the last time period.

However, the drugs did not differ at the first time period,

when it was predicted that they should.

From the sheer magnitude of the similarities here

presented between threshold effects and sleep latency

effects, it could be predicted that latency and threshold

would be highly correlated within subject. If those

correlations are done in the present data set, all except

one are significant within-subject (the one nonsignificant

correlation was at the .07 level). If those within-subject

correlations are transformed to z-scores, averaged, and

transformed back to a correlation, that average correlation

is -.39, which for the average of 46 observations per

subject, is significant at the .01 level. It does appear

that when a subject is deeply asleep and is awakened, he









can fall asleep more quickly (sleep stage controlled) than

when he is not so deeply asleep. This, of course, does not

prove a causative mechanism'but does suggest the possibility

that drugs (and various other sources) might allow subjects

to fall asleep more quickly through a common central

mechanism which also controls how deeply a person sleeps.

Such a view is consonant within most any theory of arousal.

Espoused similarity to the earlier set of results is

important at another level. The bulk of results presented

before this appendix concerned only data from the first

replication. The decision rule had been to average data to

compare it with the caffeine data in all cases, but that

rule was limited by occasional significant replication

effects. The result was that the data base for the thresh-

old results was different from that of the sleep latency

results. It could be argued that the sleep latency data,

although without a replications effect, also suffer from

whatever caused the threshold effect. This would imply

that only data from the first replication should be used

in the latency analyses. But, as might be guessed from the

lack of replication effect, if- such an analysis is done,

the results are essentially the same as using the entire

data set. It might also be added that a continued problem

in many studies of arousal is lack of agreement among

measures and concurrent definitional problems.

A correlation averaging technique was also done with

latency and sleep depth in the data from Experiment 1. The









average correlation was -.23, which was not significant.

Besides chance, this difference might be due to fewer

observations with less variance on the baseline than on the

drug nights. The possibility that the correlation in

Experiment 2 data is caused, not magnified, by drugs is

mediated against by other analyses to be reported more

fully in Appendix C. Briefly in an analysis comparing

placebo and baseline nights of deep sleep to placebo and

baseline nights of light sleep within-subject, there were

significant differences in both sleep latency questions on

the Post-sleep Inventory. Even with seven subjects, both

initial latency to sleep onset and the length of awakenings

during the night were subjectively described as signifi-

cantly shorter (p < .05) on nights of deep sleep than on

nights of light sleep. A test of actual initial sleep

latency on these nights found that six of the seven sub-

jects had shorter initial latencies on the high threshold

night, which is nonsignificant with a sign test (p = .0625)

although close. The single reversal was by two minutes.
















APPENDIX B

THE EFFECTS OF DRUGS ON TWO SUBJECTIVE
REPORT MEASURES


There is a fairly large literature on the subjective

effects of various drugs on the sleep process. It is

reviewed by Jick (1969). The present study does not seek

to deal with that topic in general but rather with two

subtopics directly related to other areas of the present

paper.

Two primary subjective measures were used in the

present study. They were the Post-sleep Inventory and the

Nowlis Mood Scale. The Post-sleep Inventory is in~develop-

mental stages (Webb, Bonnet, & Blume, 1976; Note 2,

Note 8). Sensitivity to drug conditions would help

validate that scale. Conversely, items on that scale such

as evening sleepiness and ease of awakening in the morning

could help bolster conclusions concerning the differentia-

tion of drug time course effects by threshold determination.


Method and Results


On each morning of Experiment 2 subjects were finally

awakened between 7:30 and 8:00 a.m. During the next 30

minutes they had electrodes removed and filled out the

Post-sleep Inventory and the Nowlis Mood Scale.









Responses on 29 individual Post-sleep Inventory items,

the six factors derived from that scale and six subscales

(aggression, anxiety, elation, fatigue, vigor, and

deactivation) from the Nowlis Mood Scale were analyzed in

individual subject by condition (placebo, flurazepam, and

pentobarbital) by replication analyses of variance. While

there were main effects for condition and replication in

several items, no interaction effects were found.

Therefore the data for the replicated drug conditions

were averaged and an analysis of variance with the caffeine

data was run. The analysis had effects for subjects, drug

condition (placebo, flurazepam, pentobarbital and caffeine)

and error.

Drug conditions were differentiated significantly by

eight individual Post-sleep Inventory items and three

composite factors. Of these 11 items the caffeine night

played the primary divergent role in six. The caffeine

condition was accompanied by reports of longer latency,

more evening thoughts, a more uncomfortable room tempera-

ture and lighter sleep than in any other condition. Addi-

tionally subjects felt less exhausted in the evening and

awoke more easily in the morning after caffeine than after

the other drugs.

Flurazepam administration led to reports that subjects

were significantly more sleepy at bedtime than in any other

condition. Subjects also reported higher sleepy values on

the "sleepy PM" factor, which includes exhaustion and sleep









latency, on flurazepam nights than on caffeine or placebo

nights. Also the total scale score, indicating "good"

sleep was higher for flurazepam than for caffeine. Both

flurazepam and pentobarbital nights were accompanied by

higher sleep factor scores than placebo. This composite

includes latency, awakening length, body movement and depth

of sleep.

No differences for any condition were found on the

scales from the Nowlis Mood Scale.


Discussion


In the Post-sleep Inventory data, it was the caffeine

night which was in general the clearly divergent night.

This finding is consonant with the fact that subjects were

chosen who were good sleepers. The law of initial values

would state that it would be difficult to improve the sleep

of good sleepers over baseline on any dimension but that it

would be relatively easier to disrupt it.

Clearly flurazepam and pentobarbital were found to

differ from placebo on few measures. It was claimed by

subjects that awakenings were shorter than placebo with

both drugs (corroborated in Appendix A with latency meas-

ures) and that both drugs improved sleep process factors

such as latency, body movement and sleep depth. In one

item, evening sleepiness, flurazepam was differentiated

from all other conditions including pentobarbital. This

last finding serves as subjective evidence that onset of










flurazepam activity was faster than that of pentobarbital.

Similar results were found in the evening sleepiness

factor.

The second conclusion about flurazepam from Experi-

ment 2 was that flurazepam continued to act longer in the

morning than did pentobarbital. Of the two specific

questions to assess that issue, one .(woke up extremely

tired) did not differentiate conditions. The other (had

a very hard time awakening) differentiated only caffeine

from the other drug conditions. The report was that

flurazepam made it (nodsignificantly) harder to wake up

in the morning than pentobarbital or placebo. The differ-

ence from placebo narrowly missed significance. Still, it

cannot be concluded from this data that the time course of

flurazepam continued beyond the sleep period. However, at

least one other study (Itil et al., 1974) has found

flurazepam to induce more difficulty in becoming fully

alert in the morning than did placebo.

Of the four recent studies which have examined the

effects of drugs on mood in normal subjects, only a non-

sleep study looking at mood an- hour after drug administra-

tion (Sambrooks, MacCulloch, & Rooney, 1975) has found

significant mood shifts as a result of nitrazepam and

flurazepam administration. Of studies testing morning mood

one (Schwartz, Roth, Kramer, & Hlasny, 1974) found no

effects after triazolam administration and one using

averages from three consecutive drug nights (Kales,






93


Malmstrom, Kee, Kales, & Tan, 1969) found largely only

trend effects from REMY suppressants and little effect on

mood with chloral hydrate, Qhich did not affect REM. In

light of these results, it was not surprising that no clear

trends for mood shift existed in the present, one-night

data.




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