Forced activity alters sleep cycle periodicity
and dorsal raphe discharge rhythm
R. LYDIC, R. W. McCARLEY, AND
Laboratory of Neurophysiology, Department
Boston, Massachusetts 02115
LYDIC, R., R. W. MCCARLEY, AND J. A. HOBSON. Forced
activity alters sleep cycle periodicity and dorsal raphe discharge
rhythm. Am. J. Physiol. 247 (Regulatory Integrative Comp.
Physiol. 16): R135-R145, 1984.~Single-cell activity recorded
from the brain stem dorsal raphe nucleus (DRN) of cats showed
a regular firing pattern during wakefulness (2-3 spikes/s),
decreased activity during slow-wave or synchronized sleep (1
spike/s), and cessation of regular discharge during desynchro-
nized sleep (0.05 spikes/s). DRN discharge was negatively
correlated with the onset and maintenance of the polycyclic
desynchronized sleep rhythm. These findings were derived from
analyses of DRN firing rate sampled during different behavioral
,states. The present time-course analyses of DRN discharge
described for the first time the period, amplitude, and phase
characteristics of DRN activity relative to the timing of behav-
ioral states that comprise the complete sleep cycle. The time
course of both DRN discharge and the occurrence of wakeful-
ness, slow-wave (S) sleep, transition, and desynchronized (D)
sleep were behaviorally manipulated. Forced locomotor activity
imposed by a treadmill task shortened the period length of the
sleep cycle and the duration of the DRN discharge cycle. The
magnitude and direction of these shifts in period length were
consistent across multiple sleep cycles, and there was a high
degree of coherence between the period length of the sleep cycle
and the DRN discharge profiles. The DRN discharge rate also
displayed phase dependence within S and D sleep. These results
support the hypothesis that the DRN has a physiological role
in regulating the timing of the ultradian sleep cycle.
cat; ultradian rhythms; time-normalized cycle averaging
MAMMALIAN SLEEP occurs in two phases distinguishable
by the electroencephalogram: slow-wave or synchronized
(S) sleep, characterized by slow-frequency, high-voltage
electroencephalogram waves; and desynchronized (D)
sleep, characterized by fast-frequency, low-voltage elec-
troencephalogram waves. After the termination of wake-
fulness, these two phases of sleep appear in an ordered
sequence and with periodic regularity throughout the
sleep interval. D sleep occurs as an ultradian rhythm in
most mammals and displays reliable period lengths (7)
in consolidated sleepers, such as humans (mean y = 90
-min) (4), as well as polycyclic sleepers, such as cats (mean
7 = 22 min) (36).
Putatively serotonergic neurons in the dorsal raphe
nucleus (DRN) of the cat have discharge profiles that
are related inversely to the onset and maintenance of D
sleep (22, 33). The negative selectivity of DRN neuronal
discharge during D sleep (D off) has led to general and
0363-6119/84 $1.50 Copyright 0 1984 the American Physiological
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J. A. HOBSON
of Psychiatry, Harvard Medical School,
specific hypotheses: the DRN may actively modulate (12,
14) a number of specific physiological events during
wakefulness and have a permissive disinhibitory role in
generating physiological concomitants of D sleep, such
as muscular atonia (34) or pontogeniculooccipital (PGO)
waves (22). In addition to controlling specific physiolog-
ical traits of D sleep, DRN cells may represent compo-
nents of a distributed central pattern generator involved
in initiating and maintaining the ultradian oscillation
between S and D sleep states, as postulated by the
reciprocal interaction model of sleep cycle control (13,
21). These physiological trait-vs.-behavioral state hy-
potheses concerning DRN function are not mutually
exclusive. Pharmacological (32) and surgical (29) manip-
ulations of the DRN have been used to determine the
degree to which DRN discharge is globally related to
behavioral states and/or specific physiological variables
comprising the states.
Another way to examine the contribution of DRN to
control of behavioral state and incidental state signs is
to manipulate the behavior of intact and unanesthetized
animals from which DRN cells can be recorded. This
paper describes experiments designed to induce major
shifts in the temporal distribution of sleep states while
monitoring the discharge of single cells in the DRN of
intact spontaneously sleeping cats. Using forced loco-
motor activity to manipulate the sleep cycle, this study
examined the null hypothesis that there is no consistent
relationship between the sleep cycle and the DRN dis-
charge cycle. Although forced activity significantly al-
tered the periodic properties of the ultradian sleep cycle,
the sleep cycle could not be dissociated from the DRN
discharge cycle. The high degree of coherence between
the period length of DRN discharge and the timing of
the sleep cycle supports the alternate hypothesis that the
temporal characteristics of sleep are tightly linked to the
time course of DRN discharge. The results of these time-
course analyses are consistent with the hypothesis that
DRN is a control element involved in regulating the
ultradian sleep cycle.
METHODS
Neurophysiological and Behavioral
Nine adult male cats were chronically implanted with
electrodes for recording electroencephalogram (EEG),
electroocculogram (EOG), and nuchal electromyogram
Society R135
R136
(EMG). The electrographic data obtained from these
electrodes were used for conventional scoring (36) of
sleep and wakefulness. For extracellular recordings, two
sets of microwire bundles, each comprised of six 62-grn-
diam and three 32-lrn-diam wires, were aimed for im-
plantation in the DRN (P = -1.5, L = 0.0, H = 1.0, f) =
45” posterior). Microwire electrodes provided stable re-
cordings of single-cell activity in behaving animals. As
detailed elsewhere (9), the microwires were inserted into
guide tubes, which are part of a miniature microdrive
assembly. The tips of the stainless steel guide tubes were
stereotaxically aimed at a point several millimeters above
the DRN target site. The microdrive was permanently
attached to the skull with dental acrylic; the microwire
bundles were then inserted into the guide tubes to a
depth of about 1 mm above the DRN. The microwire
bundles were held at this position by gluing them to the
guide tubes. Each of the 18 microwires was soldered to
an electrical connecting plug, which was also chronically
implanted on the skull. Each cat was then allowed 2 or
3 wk to recover from the surgery before recording ses-
sions began. During the recordings, the 18 microwire
electrodes were advanced along the dorsal-to-ventral
plane by turning an 080 machine screw on the micro-
drive. The cats were maintained under constant illumi-
nation in their home cages as well as in the recording
apparatus.
On recovery from the surgery and immediately preced-
ing the single-unit and electrographic recording session,
the cats were exposed to one of two behavioral condi-
A B -
Minufes
c cycles (e.g., cycle 1 and cycle 4) are chosen on basis of
28
24
0 1- -I i--L.--
oi- 20 40 60 80 100
% Cycle Completed
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LYDIC, McCARLEY, AND HOBSON
tions: 1) forced activity in which cats spent 12-16 h in a
treadmill that moved at 0.05 m/s or 2) ad libitum waking
and sleeping in which cats spent an equivalent amount
of time in their home cage. After either of these two
conditions, the cats were placed in an electrically
shielded chamber, and recordings were obtained through
a standard commutator-swivel system. Once attached to
the cable and swivel, cats were either free ranging or
head restrained within the shielded chamber. When the
data from head-restrained and free-ranging recording
paradigms were systematically compared, there was no
significant difference in the time course of the D-off cell
activity curves or the period lengths of the sleep cycle.
Time-Normalized Sleep Cycle Averaging
The period length of the sleep cycle was defined as the
amount of time from the end of the D sleep episode in
one cycle through the end of D sleep in the subsequent
sleep cycle. Each sleep cycle was comprised of a regular
progression of behavioral states. In the cat, D sleep is
typically followed by waking (W), then S sleep occurs
and is followed by a transition state (T) (operationally
defined here by the occurrence of eight or more PGO
waves per minute), which heralds the onset of another D
sleep episode.
In spite of this orderly progression of sleep cycle com-
ponent states (W, S, T, D), the ultradian D sleep rhythm
in the cat has been noted (35, 36) to vary by amounts
ranging from 5 to 40 min. Although it was appropriate
Cyde 4
1
FIG. 1. Schematic representation of time-normal-
ized sleep cycle-averaging technique. A: individual
established criteria from a series of cycles. B: cycles I
Cycle f und Cycle 4 and 4 have similar amplitudes but different period
lengths. C: period length in minutes is normalized to
100%. For a cycle where T = 20 min, each of 10
normalized bins (abscissa) would reflect 2 min of neu-
ronal activity. Total discharge activity in cycle is
calculated and amplitude of histogram at each of 10
bins represents % of this total activity (ordinate). D:
once individual cycles are time normalized, they are
arithmetically averaged. Additional details are pro-
vided in text.
DORSAL RAPHE DISCHARGE RHYTHMS Hl%

to consider the sleep cycle as a rhythmic unit, the vari- 20 min could not be averaged with neuronal activity
ability in period length precluded arithmetic averaging associated with a 25-min cycle. Rather the phase of the
across cycles of physiological or behavioral events that separate behavioral states and the relationship between
are specifically related to sleep cycle phase. For example, the sleep cycle and simultaneously recorded neuronal
neuronal activity associated with a sleep cycle where T = activity had to be considered.
It is, however, possible to average any dependent mea-
sure across multiple sleep cycles if the period length of
each cycle is normalized with respect to duration. This
process of time normalization transforms the period
length in minutes into a standardized length expressed
as a percentage of the cycle completed (Fig. 1).
Time-normalized sleep cycle averaging of DRN activ-
ity involved a four-step analytic process. I) The poly-
graphic records were scored at l-min bin intervals for
behavioral state (W, S, T, or D), and the DRN discharge
rates were tabulated during each of these 1-min intervals.
2) Cycles to be time normalized (Fig. 1A) were selected
according to criteria of period length and the presence of
normal state progression. 3) Individual cycles (Fig. 1B)
FIG. 2. Cellular discharge of dorsal raphe nucleus cell during wake- were expressed as the number of DRN discharges per
fulness. Calibration bars: horizontal, 1.0 s; vertical, 200 PV. second (ordinate) during each minute of polygraphic

NC:. 3. I’hotomicrograph of midsagittal view of brain stem of cat. This section, roughly corresponding t,o laterality 0.2, shows lesion (arrour)
made by microwire electrode within dorsal raphe nucleus. Calibration bar, 1 mm.

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R138 LYDIC, McCARLEY, AND HOBSON

recording. The period length of the cycle (abscissa) was
converted from time in minutes into 10 standardized
bins (Fig. 1C). These normalized bins may be read as a
percent of the cycle completed and range from 0 to 100.
The corresponding DRN discharge level was converted
to spikes per second and in each normalized bin was
expressed as the percent of total DRN spikes in the cycle.
4) Additional cycles were transformed in a similar man-
ner, and once normalized any number of cycles could be
summed and averaged (Fig. 1D). Although the sleep cycle
period lengths vary, the cellular activity associated with
each cycle received equal arithmetic weighting, since
discharge level per bin was expressed as a percent of
total spikes per cycle.
The variability of the ultradian D sleep rhythm has
been noted (35,36), and in the present study sleep cycles
were selected for time-normalized averaging according to
period length. Sleep cycles, each defined from D sleep
end to D sleep end, were categorized for time normali-
zation and averaging into four groups on the basis of
period length. Sleep cycles were defined operationally as
short (T < 15 min) and usually involving an abortive D
sleep episode, regular cycles (T = 15-30 min), long cycles
(7 = 31-45 min), and extended cycles with period lengths
in excess of 45 min.
The present results were derived from sleep cycles
with period lengths ranging from 15 to 30 min (regular
cycles) and from cycles where 7 ranged from 31 to 45
min (long cycles). All time-normalized sleep cycle aver-
aging occurred within one of these two groups, and in no
case were regular and long cycles averaged together.
RESULTS
Characteristics of Spontaneous DRN Discharge
Long-term recordings of single-cell activity were ob-

,,,,,,,,; 1 . .T
., , /‘/,.I’ \ “l,‘,
’ 1 ’ /

UNIT/10
-

EOG

FIG. 4. State-related changes in dorsal raphe nucleus (DRN) activ-
ity and relationship between DRN discharge and polygraphic variables
used operationally to define behavioral states. A: wakefulness changing
to slow-wave sleep. B: slow-wave sleep and transition characterized by
increased pontogeniculooccipital (PGO) wave activity and decreased
DRN tiring. C: state change from transition to desynchronized sleep.
D: termination of desynchronized sleep and resumption of wakefulness.
EMG, electromyogram; EEG, electroencephalogram; unit, Schmitt trig-
ger output of DRN activity; unit/lo, Schmitt trigger output divided by
10; EOG, electrooculogram. Time mark above EOG trace indicates 1
min.

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DORSAL RAPHE DISCHARGE RHYTHMS R139
tained from nine D-off cells across 300 complete sleep TABLE 1. Behavioral state and dorsal raphe
cycles. All spike trains, such as those illustrated in Fig. single-unit activity as a function of 7
2, displayed three characteristics indicative of stable
recordings. 1) The amplitude and waveform of the re- Behavioral State
corded potentials were similar throughout the recordings. Wake- s sleep Transi-
D sleep
2) The state-related firing rates (W-to-D selectivity ra- fidness tion
tios) were similar from cycle to cycle. 3) DRN unit Regular cycles (7 = 25.2 ,t 2.8 min)
activity was consistently related to specific phases of the, Percent of time 4.9 35.4 17.0 42.7
sleep cycle. After the experiments, the individual micro- (F = 27.9; df = 3,40; k6.1 t10.8 k8.0 k15.8
wires from which a D-off cell had been recorded were P < 0.001)
histologically localized (Fig. 3) by passing a direct current Dorsal raphe discharge 1.6 1.0 0.45 0.17
rate, spikes/s
(20 PA for 10 s) to mark the recording site with a small (F = 85.6; df = 3,34; kO.31 to.16 _tO. 16 *0.09
lesion. P < 0.001)
The state-specific firing pattern of DRN cells (Fig. 4
Long cycles (7 = 38.3 t 4.2 min)
and Table 1) has been characterized elsewhere (12, 22,
Percent of time 22.5 43.2 8.0 26.3
33) as maximal during W (2-3 spikes/s), slowing during (F = 28.8; df = 3,56; k13.1 HZ.8 *4.9 k8.8
S and T (5 1 spike/s), and stopping during D sleep. One- P < 0.001)
way analyses of variance revealed statistically significant Dorsal raphe discharge 1.2 0.69 0.34 0.03
differences in dorsal raphe discharge rate across these rate, spikes/s
four behavioral states during both the regular- (P < (F = 47.6; df = 3,55; kO.41 zko.30 *0.19 *0.03
P c 0.001)
0.001) and long- (P C 0.001) duration sleep cycles (Table
1). Figure 4 illustrates the relationships between DRN Values are means Z!ZSD. 7, cycle period length. Sleep cycle of cats is
comprised of 4 polygraphically defined behavioral states: wakefulness
activity and specific physiological variables used for be- (W), synchronized (S) sleep, transition (T), and desynchronized (D)
havioral state classifications. Particularly striking is the sleep. These behavioral states are expressed in an ordered sequence
negative correlation between DRN firing and PGO waves and occur rhythmically with period lengths in &radian range. Individ-
and between DRN firing and nuchal muscle tone. The ual sleep cycles were defined from end of D sleep in 1 cycle to end of
samples of behavioral states shown by Fig. 4 do not D sleep in subsequent cycle. Regular 7 cycles (n = 11) had period
lengths ranging from 15 to 30 min. Long r cycles (n = 15) ranged from
sufficiently emphasize that behavioral states are contin- 31 to 45 min in length. Within each of these 2 groupings based on 7,
uous and that state transitions do not occur as step one-way analyses of variance revealed a statistically significant behav-
functions. Figure 4 clarifies the sampling problems in- ioral state effect (F) for 2 dependent variables: 1) percent of time spent
herent in such static snapshot visualizations of neuronal in each state and 2) dorsal raphe (DRN) single-unit discharge rate.
Independent t test comparisons of regular and long cycles revealed
activity, since each frame can show only a small portion statistically significant differences in percent of time spent in W (t =
of a complete sleep cycle. To address the precise phase 4.14, df = 24, P < O.OOl), T (t = 3.6, df = 24, P < O.OOZ),and D sleep
relationships between DRN discharge, behavioral states, (t = 3.4, df = 24, P < 0.005). Similar comparisons for S sleep revealed
and specific state variables, such as PGO waves, numeric no statistically significant differences. Period length in minutes of
or conceptual representations of neuronal activity across regular and long sleep cycles was significantly’ different (t = 8.9, df =
24, P < 0.001).
time require data that represent the entire sleep-wake
cycle. dorsal raphe discharge rhythm. The DRN discharge pro-
files typically had longer 7 after no forced activity (Fig.
Forced Activity Alters Period Length 5A) and shorter 7 after forced activity (Fig. 5B). Table
of Dorsal Raphe Discharge
1 summarizes the average differences in DRN discharge
Prolonged periods of forced wakefulness or locomotor rate observed in association with the regular (15-30 min)
activity immediately before the recording session dra- and long (31-46 min) 7 sleep cycles. Cosinor spectral
matically altered the neuronal discharge profiles re- analysis of a typical 5-h sample of DRN discharge re-
corded from DRN cells (Fig. 5, A vs. B). Forced activity corded after forced activity revealed (Fig. 6) a dominant
tended to regularize the time course of DRN discharge period length in the 15. to 20-min range. This period was
(Fig. 5B). Figure 5A illustrates the discharge pattern significantly different (P < O.OOl), by the least-squares
from the same D-off cell that generated the profile in method (30) of wave fitting, from the null hypothesis
Fig. 5B. However, for 12-16 h before the recording ses- that the amplitude of the D sleep rhythm was zero. The
sion, the cat remained in its home cage and was not ? value associated with the 17-min period indicated that
subjected to the forced activity paradigm. With no forced a sine wave of this duration accounted for the largest
activity, the subsequently recorded DRN single-unit dis- portion of the variance displayed by 300 min of DRN
charge profiles (Fig. 5A) were comprised of cycles with activity. When no forced locomotor activity preceded a
longer period lengths. The observed relationships be- 6-h and 7-min DRN recording, this same cell discharged
tween period length of DRN discharge and the preceding with a dominant period length of about 37 min (P <
behavioral condition does not imply exclusivity. Regular 0.001). The ? value indicated that the best-fitting sine
period length cycles do occur, though less frequently, wave of 36.5 min accounted for the largest degree of
after no forced activity. variance in the 367 min of DRN recording. When these
The two behavioral conditions preceding the recording data (Fig. 5, A and B ) were detrended, similar results
sessions altered the ultradian period length (7) of the concerning period length of the ultradian DRN discharge

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 R140 LYDIC, McCARLEY, AND HOBSON

FIG. 5. Single-cell activity profiles recorded from dorsal raphe nu- an activity wheel. Ordinate gives spikes/s; abscissa gives time in min.
cleus after 2 different behavioral treatments. A: DRN activity profile Black bars indicate periods of desynchronized sleep. Open circles denote
recorded from a cat that had spent preceding 12 h in a standard cage. periods of wakefulness.
B: DRN activity recorded from a cat that had spent preceding 12 h in

FwcT,kAkity first half of the averaged sleep cycle, the cat spent most
j =
of the time in S sleep (Fig. 7A2). However, throughout
the progressing sleep cycle the time spent in S declined
and the time spent in T increased (Fig. 7A3), reaching
maximal levels by 60% sleep cycle completion. From 0
to 60% of the cycle (abscissa) DRN activity showed a
steady decline. During the final 40% of the cycle (70-
100% cycle completion), D sleep (Fig. 7A4) accounted
for the majority of the state distribution, and DRN
discharge declined to its lowest level.
Figure 7, Bl-B5, shows the time course of DRN dis-
charge and simultaneously measured behavioral state
FIG. 6. Time series analysis period spectrum of dorsal raphe nucleus sequence, but these data were obtained from sleep cycles
(DRN) discharge. Function outlined by closed circles shows spectral with long period lengths. Note that for each of the
components of DRN rhythm following 12-16 h of forced locomotor
activity imposed by treadmill task. Open circles show DRN rhythm component behavioral states (W, S, T, and D) the pro-
recorded from same cell when cat spent preceding 16 h in its home cage files of the long cycles are shifted to the right and all
(no forced activity). Peaks of most dominant period (7) are indicated states extend into later phases of the sleep cycle. Long
for each behavioral condition. These results were obtained from least- cycles also revealed a significantly greater (P < 0.001)
squares-based time series analyses (30). percent of time spent in wakefulness (22.5%) than did
the regular cycles (W = 4.9%). Although the state profiles
rhythms were obtained from time series analyses based for wakefulness are strikingly different for the regular
on the BMDP time series analysis (PlT), which uses and long period-length cycles (Fig. 7, Al vs. BZ ), the
DRN discharge profiles during the first half of the sleep
Fourier transform techniques (5).
cycle were very similar (Fig. 7, A5 and B5). A larger
percent of time was spent in S sleep during the long
Sleep Cycle Duration, Time Course of Behavioral
cycles (43.2%) than during the regular cycles (35.4%),
States, and Dorsal Raphe Discharge but these differences were not statistically significant
Figure 7 illustrates the time-normalized multiple sleep (Fig. 7, A2 and B2, and Table 1). During the long sleep
cycle average of DRN discharge and behavioral- state cycles, 8% of the time was spent in T as compared with
associated with the regular period-length cycles (7 = 25.3 17% of the time during the regular 7 cycles, and these
t - 2.7 min) and for sleep cycles with long period lengths differences in T were statistically significant (P < 0.002).
( = 38.3 $- 4.2 min). The duration in minutes of the D sleep tended to occur at later portions of the average
Ggular and long sleep cycles differed significantly (t = cycle (Fig. 7B4) when sleep cycle 7 was increased. The
8.9, df = 24, P < 0.001). percent of time in D sleep during the long cycles (26.3%)
For the regular cycles (Fig. 7, Al-A@ waking was was significantly (P < 0.005) less than with the regular
maximal during the initial 10% of the sleep cycle (Fig. cycles (D = 42.7%). DRN firing levels were increased
7Al) as was the DRN discharge (Fig. 7A5). As early as slightly in association with the long sleep cycles (Fig.
20% into the sleep cycle, both DRN discharge and the 7B5); the D-off firing pattern was not as pronounced,
amount of time spent in W began to decline. During the and the minima of neuronal discharge occurred later in

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DORSAL RAPHE DISCHARGE RHYTHMS R141
PEGULAR CYCLE: , LONG CYCLES , individual states comprising the sleep cycle (Table 1)
were in part a function of the behavioral conditions
immediately preceding the recording session. During
both the regular and long cycles, DRN activity was
60 60 maximal in the initial 10% of the sleep cycle.
The T profiles (Fig. 7, A3 vs. B3) were similar during
long and regular cycles. For both the regular and long
cycles, DRN unit activity accounted for about 12% of
’ 0 20 40 60 80 IO0
the total neuronal discharge when T was maximal.
The time course of D sleep (Fig. 7, A4 and B4) dis-
00-1
played similar phase positions during both the regular
80 and long cycles. DRN discharge associated with the
60 regular sleep cycles (Fig. 7A5) increased during the final
40 portion of the cycle. This increased DRN discharge was
less marked in association with long-period sleep cycles
20
(Fig. 7B5).
’
Phase Dependence of Dorsal Raphe Discharge
T within Slow- Wave and Desynchronized Sleep
80
The previous section has shown the strong state-de-
60 pendent discharge of DRN across the behavioral states
comprising the ultradian sleep cycle. The time-normal-
ized multiple sleep cycle averages of DRN discharge (Fig.
7) strongly suggested a continuous modulation of firing
0
0 20 40 60 80 100 rate rather than an abrupt or discontinuous alteration in
DRN discharge associated with changes in behavioral
state. If DRN discharge is continuously modulated across
the sleep cycle, such changes in firing rate should be seen
within S or D sleep. This possibility was examined quan-
titatively by characterizing the phase dependence of
DRN firing rate within S and D sleep. To evaluate any
discharge rate changes within sleep states, linear regres-
0 20 40 60 80 100 sion analyses were performed using DRN discharge rate
in spikes per second as the dependent variable (y) and
2 22 time in minutes within a single behavioral state (S or D
.% 18 sleep) as the independent variable (x).
@ 14 Synchronized sleep. During S sleep associated with the
$ to IO regular-7 cycles, the average slope (spikes per second
t$ 6 6 across each minute of the state) was -0.02, with 10 of 11
& 2 2 individual cycles showing a negative slope. During S
sleep, 10 of 15 long 7 cycles revealed declining discharge
20 40 60 80 100 0-o
rates and an average slope of -0.02. For precise statistical
% Cyde Complefed evaluation of these DRN discharge rate changes, the S
FIG. 7. Histograms illustrating time course of state components (W, sleep epoch of each sleep cycle was divided into three
S, T, D, waking, slow-wave, transition, and desynchronized, respec-
tively) comprising sleep cycle and time course of simultaneously re-
phases: early, middle, and late. The DRN discharge rate
corded dorsal raphe neuron (DRN) activity. Frames Al-A5 were ob- was then rank ordered (3 = maximum rate to 1 =
tained from 11 sleep cycles (7 25 min) with period lengths ranging minimum rate) during each of these three phases of
from 15 to 30 min. Frames BI-B5 were derived from 15 sleep cycles synchronized sleep. This ranking procedure revealed a
(7 38 min) with period lengths ranging from 31 to 46 min. For % state monotonically declining discharge rate (a rank ordering
histograms, each portion of cycle completed sums to 100 across 4 states.
For example, reading 10% cycle completed bin from Al to A4 indicates of 3,2,1) in 10 of 15 long-7 cycles and in 6 of 11 regular
that W accounted for about 20% of state distribution, S for 70%, T for 7 cycles. The multinomial probability (P) of obtaining a
about lo%, and 0% of time was spent in D. For DRN histograms (A5 3,2,1 ranking vs. the null hypothesis of observing this
and 85), neuronal activity at each portion of cycle completed (abscissa) ranking by chance alone was calculated. The observed P
is expressed as % total spikes (ordinate).
< 0.01 for the regular-7 cycles and P < 0.001 for the
long-T cycles supported the hypothesis that during S
the sleep cycle. sleep the DRN displayed a phase-dependent decrease in
Comparisons within the regular 7 (Fig. 7, Al-A5) or discharge rate, irrespective of sleep cycle T.
long T (Fig. 7, BI-B5) cycles reveal, in further detail, the Desynchronized sleep. Similar linear regression anal-
relationship between the DRN discharge profiles and the yses revealed an increasing DRN discharge rate from
time course of specific behavioral states comprising the early to later portions of D sleep.
sleep cycle. Both the neuronal activity profiles and the For the regular--r cycles, the average slope was +0.02

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R142
with 8 of 11 cycles showing a positive slope. The time-
normalized average (Fig. 7A5) indicated an increased
DRN discharge rate during the final portion (90-100%
cycle completed) of the regular-T cycles. The binomial
theorem was used to calculate the probability that the
DRN discharge rate during the final third of -each indi-
vidual sleep cycle was greater than the mean firing rate
during the preceding two-thirds of each cycle. The result
of these calculations (P = 0.3) did not achieve statistical
significance, although the trend of increased firing during
the final bin was present in 7 of 11 regular-7 cycles.
Only 10 of 15 long-7 cycles displayed any DRN dis-
charge during D sleep. For these 10 long-7 cycles, the
average slope was +0.02, and 9 of 10 individual cycles
had a positive slope. For the long-7 time-normalized cycle
(Fig. 7B5), 9 of 10 individual sleep cycles displayed the
greatest DRN discharge rate during the final third of the
D sleep epoch. Again the binomial theorem was used to
calculate the exact probability that the DRN firing rate
during the final thi rd of each D sleep epoch was greater
than the mean rate during the preceding two-thirds of
each D sleep epoch. The probability of this ordering (P
= 0.011) supported the hypothesis of a statistically sig-
nificant phase-dependent increase in DRN discharge
during the D sleep epochs associated with the long-7
sleep cycles.
DISCUSSION
Phase Relationship Between DRN Discharge
and Behavioral State
Global classifications of behavioral state (W, S, T, or
D) are traditional and offer a useful scheme for studying
cellular elements or physiological systems during sleep
and wakefulness. However, the precise phase relation-
ships between DRN discharge and physiological or be-
havioral events comprising the sleep cycle may not be
readily apparent when DRN activity is averaged accord-
ing to discontinuous classifications of behavioral state
(Table 1). The present time-course data, derived from
the cycle-averaging technique, provided phase-aligned
comparisons between DRN discharge profiles and con-
current behavioral state fluctuations (Fig. 7). Such com-
parisons using intact unanesthetized animals are critical
for examining the temporal relationships between DRN
discharge and behavioral states comprising the ultradian
sleep cycle.
For example, a distinctive finding that emerged from
the present approach is that DRN discharge shows phase
dependence within S and D sleep. Furthermore it appears
that the period length of the sleep cycle also influences
the profile of the DRN discharge (Fig. 7, A5 W. B5).
Within S sleep, DRN discharge exhibited a statistically
significant decrease (mean slope = -0.02 spikes s-l
min-‘) for both the regular- and long-7 sleep cycles.
During desynchronized sleep, regression analyses re-
vealed an increased DRN firing rate (mean slope =
+0.02) for both long- and regular-7 sleep cycles. This
phase-dependent increase was statistically significant for
the long-7 cycles. For the regular-7 cycles, the trend of
increasing DRN discharge was present during the D sleep
Downloaded from journals.physiology.org/journal/ajpregu at UNAM Fac De Med Dir Gen De Biblio (132.248.142.040) on February 10, 2023.
LYDIC, McCARLEY, AND HOBSON
epochs of most (7 of 11) individual cycles. The regular-7
cycles exhibited a 177% average increase in DRN firing
rate within D sleep (Fig. 7A5,90-100% cycle completed),
and the burst of DRN activity before the termination of
D sleep was present in the majority of these recordings.
The finding that the conservative nonparametric statis-
tic did not achieve a P < 0.05 significance level may be
a function of the small sample size, and this possibility
can be examined by future analyses of a larger sample of
regular-7 cycles. Considered together, the results of the
linear regression analyses, the outcome of the probability
calculations, and the observations by others (18, 33) of
increased DRN firing near sleep cycle end, all demon-
strate the presence of phase-dependent DRN discharge
within (Fig. 7) and between (Table 1) behavioral states
comprising the ultradian sleep cycle. These findings em-
phasize that the precise temporal phase of the sleep cycle
must be considered when characterizing the relationship
between DRN activity and the onset, maintenance, or
offset of a behavioral state.
DRN as a Putative Neuronal Regulator
Considering only the dorsal nucleus of the extensive
raphe complex, a large body of literature suggests that
the DRN is involved in regulating multiple physiological
systems. For example, the DRN has been shown to be of
functional importance for the regulation of ovulation
(24) and the neuroendocrine control of progesterone (37)
and luteinizing hormone (1) secretion, to exert pressor
effects on the cardiovascular system (40), and to mediate
stimulation-produced analgesia (28) and the function of
endogenous nociceptive systems (31).
Neurophysiological studies investigating the cellular
basis of sleep cycle control have long associated levels of
DRN activity with behavioral arousal (33), the onset of
S sleep (3), and/or the ultradian D sleep rhythm (22).
More recently, serotonergic input to the hypothalamic
suprachiasmatic nuclei from the midbrain raphe has been
suggested (39) to be of potential importance for the
functional organization of neural elements comprising
the circadian system (25). The diversity of these data
emphasize the utility of pluralistic concepts regarding
the functional role of the DRN. The hypothesis that the
DRN has a continuous neuromodulatory role influencing
the probability of a set of behavioral states (11, 12, 14)
is probably a more appropriate general concept than
exclusive hypotheses that DRN activity is related either
to behavioral states (i.e., causes D sleep by disinhibition)
or to single physiological traits (i.e., causes PGO waves
or motor atonia by disinhibition). Although specific func-
tional relationships may also be true, such relationships
alone fail to describe the global functional role of the
DRN.
l l Investigations (12, 22, 33) that have assessed the de-
gree of DRN involvement in behavioral state control
have relied exclusively on rate and pattern analyses. The
present time-course analyses extend these findings con-
cerning state-dependent firing of DRN by focusing on
the relationship between DRN activity and the temporal
organization of the sleep cycle. It is clear from Figs. 5
and 7 that the time course of DRN activity was strongly
DORSAL RAPHE DISCHARGE RHYTHMS
influenced by the behavio ral cond itions that preceded
the recordi .ng session. Cats exposed for 12 -16 h to forced
activity had DRN discharge profiles that were shorter
and more regular (Fig. 5) than DRN activity recorded
from cats with no forced activity before the recording
session. Thus the temporal organization (Fig. 7 and
Table 1) of both DRN discharge and the sleep-wake cycle
were predictably modified by behavioral experience that
preceded these recording sessions.
Significance of Observed Behavioral Influence
on Sleep Cycle Regularity
During the treadmill task associated with forced activ-
ity, there was no fine control over the physiological
response to the exercise challenge. While in the tread-
mill, no measures were taken of 02 consumption, core
body temperature, cardiovascular parameters, or EEG
variables. Although the precise physiological concomi-
tants of the treadmill task remain unspecified, Fig. 7
reveals that the prolonged period of forced locomotion
dramatically reduced the amount of time spent in wake-
fulness. The length of the sleep cycle is strongly corre-
lated with th .e amount of W that is present. Studies using
rats have noted that longer-7 sleep cycles were associated
with increases in W (8). In the present study, shortening
of sleep cycle period length was associated with a reduced
amount of W.
This spontaneous reduction in W after forced activity
is analogous to the sleep cycle scoring technique
(compression) in which naturally occurring epochs of W
are excluded from the behavioral state data. Sleep cycle
studies (35) with cats have noted that the compression
technique increased the goodness of fit (r value) between
autocorrelation functions of the sleep cycle and best-
fitting cosine waves. This degree of fi t was estimated
from ? values used to quantify the amount of variance
in the a utocorrelation function accounted for by damped
cosine waves. The p values were thus used to estimate
the most likely period length of the ultradian sleep cycle.
These data revealed a stronger periodicity in the ultra-
dian sleep cycle when W epochs were not included in the
analyses. These findings were interpreted as supporting
the notion that the D cycle may be a purely sleep-
dependent process rather than a manifestation of the
basic rest activity cycle (BRAC). The BRAC hypothesis
(X5), reviewed elsewhere (16, 26), hypothesized that a
single, neurophysiologically unspecified, driving oscilla-
tor might underlie all ultradian rhythms.
Alternatively, one may interpret the foregoing con-
cepts emanating from the BRAC hypothesis in light of
the present data and recent findings at the cellular-
molecular level concerning the activity of cholinergic and
aminergic neurons. The time course of DRN activity
(Figs. 5-7) does not suggest, as implied by the BRAC
hypothesis, oscillations that display a relatively fixed 7
and variable amplitude. The discharge profiles of puta-
tively aminergic DRN activity emphasized the presence
of continuous modulation (Figs. 5 and 7) rather than a
fixed discharge pattern across behavioral states (Table
1). Whenever putatively aminergic DRN neurons have
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R143
been recorded from intact unanesthetized animals, the
level of cellular discharge has been found to be positively
correlated with W and inversely related to the onset of
the ultradian sleep cycle. These observations support the
hypothesis (6,12,18,22) that the period length of the D
sleep oscillator is strongly modulated by the synaptic
activity of aminergic systems. In the present experiment,
the prolonged locomotor activity imposed by the tread-
mill condition was probably accompanied by an increase
in the amount of time spent in W (7), which in turn
abnormally prolonged the activity of DRN cells. Since
DRN cells are known to have recurrent inhibitory col-
laterals (27,38), these extended periods of DRN activity
would predict the development of a cumulative autoinhi-
bition. When the cats were removed from the activity
wheel and placed in the recording apparatus, signaling
the onset of the sleep interval, this accumulated auto-
inhibition would reduce the synaptic efficacy of afferent
input to and efferent output from the DRN. On the DRN
afferent side, this would mean DRN discharge profiles
would be more regular after forced activity (Fig. 5B) and
more noise dominated (Fig. 5A) without prior intervals
of forced activity. Consistent with these predictions,
during the sleep interval that followed forced activity,
the amount of W decreased (Table 1 and Fig. 7) and
perturbations of DRN discharge were fewer (Fig. 5).
These changes in behavioral state and DRN discharge
levels are also influenced by reticular systems (23) that
mediate arousal. In consideration of DRN efferents, pro-
longed intervals of forced W and the build up of auto-
inhibition would result in a diminution of net aminergic
output corresponding to a diminished synaptic efficacy.
This decreased aminergic modulation is hypothesized by
the reciprocal interaction model to d&inhibit putatively
cholinergic cell populations in the medial pontine retic-
ular formation (mPRF), which have firing levels posi-
tively correlated with the onset of D sleep (13, 21). One
result of the DRN and mPRF interaction postulated by
the reciprocal interaction hypothesis would be an in-
creased frequency of D sleep epochs (Fig. 5B) and an
increased duration of D sleep episodes (Fig. 7 and Table
1) following forced activity.
Dorsal Raphe Involvement
in Behavioral State Modulation
Several lines of evidence reinforce the explanatory
value for the foregoing interpretation of the present
findings. Anatomic evidence, reviewed elsewhere (19, 27,
38), supports the notion of recurrent collaterals, which
are autoinhibitory to DRN, and the idea of synaptic
contact between DRN and mPRF. For example, recent
physiological studies reported that electrical stimulation
of mPRF enhanced the discharge of DRN cells and that
this mPRF-induced facilitation of DRN activity was
absent after the administration of chloral hydrate (10)
and during the spontaneous D sleep intervals (17). Phar-
macological elicitation of a behavioral state resembling
D sleep (2) by the microinjection of carbachol, a cholin-
ergic agonist, into the mPRF is congruent with single-
unit studies (20) demonstrating that putatively cholin-
R144 LYDIC, McCARLEY, AND HOBSON

ergic mPRF neurons exhibited increased discharge rates
that were phase locked to the D sleep cycle. Finally,
behavioral evidence concerning manipulation of the
polycyclic sleep cycle suggests that forced activity de-
prives cats ofD sleep (,7). Durin .g subsequent sleep inter-
vals, cats exposed to forced activity exhibit an increased
frequency and intensity of D sleep, a phenomenon re-
ferred to as a D sleep rebound (7) (Fig. 7, A4 vs. B4, and
Table 1). If the rebound of D sleep is, in part, modulated
by a cessation of DRN discharge that is inhibitory to the
expression of D sleep, one would expect to see a reduction
in DRN activity (Fig. 7, A5 vs. B5) during the D sleep
rebound that followed forced activity. The foregoing
evidence derived from anatomic, behavioral, pharmaco-
logical, and electrophysiological studies strongly sup-
ports the hypothesis that the DRN influences the ultra-
dian D sleep rhythm.
The present data provide the first description of the
correlations between the time course of DRN single-unit
activity and the time course of behavioral states that
comprise the ultradian sleep cycle. Although these data
characterized the complete time course of behavioral
state and DRN activity profiles, such correlative evi-
dence cannot prove that DRN discharge is causally re-
lated to the ultradian sleep cycle. It can be fairly con-
eluded, however, that 1) with no forced activity the
period length of the sleep cycle and the period length of
the DRN discharge cycle were tightly linked in the intact
undrugged cat: 2) the &radian sleep cycle and the DRN
discharge rhythm always exhibited similar period
lengths, yet both were altered significantly and with a
high degree of coherence by forced activity; and 3) the
DRN discharge profiles exhibited significant phase de-
pendence across the behavioral states comprising the
ultradian sleep cycle and within S and D sleep. .These
conclusions, derived from time-course analyses, are con-
sistent with a large body of evidence obtained from DRN
discharge rate analyses (12,14,18,19,22,33). Both time-
course and discharge rate analyses support the hypoth-
esis that the DRN modulates through disinhibition many
of the physiological events associated with S and D sleep.
We thank Dr. H. Baghdoyan and Dr. P. Lavie for helpful suggestions
and Dr. S. Massaquoi and A. Strassman for computer programming.
This study was supported by National Institutes of Health Grants
MH-13923, MH-280 to R. W. McCarley, and MH-14275 to R. Lydic.
Received 11 July 1983; accepted in final form 6 February 1984.

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