Psychoneuroendocrinology 29 (2004) 669685

www.elsevier.com/locate/psyneuen

Circulating neurotransmitters during the

different wakesleep stages in normal subjects
Fuad Lechin a,b,c,, Betty Pardey-Maldonado b,c, Bertha van der
Dijs a,c, Mireya Benaim c, Scarlet Baez a,c, Beatriz Orozco c,
Alex E. Lechin d
a

Laboratories of Neurochemistry, Department of Physiological Sciences, Instituto de Medicina

Experimental, Universidad Central de Venezuela, Caracas, Venezuela
b
Sleep Research, Department of Physiological Sciences, Instituto de Medicina Experimental,
Universidad Central de Venezuela, Caracas, Venezuela
c
Section of Neuropharmacology, Department of Physiological Sciences, Instituto de Medicina
Experimental, Universidad Central de Venezuela, Caracas, Venezuela
d
Sleep Disorders Center, Memorial Herman Hospital, Houston, TX, USA
Received 10 July 2002; accepted 5 May 2003

Abstract
We investigated the changes of circulating neurotransmitters during the wakesleep cycle
in order to find possible correlations with the activity of central neurocircuitry functioning.
Noradrenaline (NA), adrenaline (Ad), dopamine (DA), platelet serotonin (p-5HT), plasma serotonin (f-5HT) and plasma tryptophan (TRP) were assessed during the morning (supine
resting + 1-min orthostasis + 5-min exercise) and at night (supine resting + slow wave
sleep (SWS) + REM sleep). Only NA increased in the plasma during short-lasting (1-min)
orthostasis morning waking period. Both NA and Ad rose during moderate exercise. The
nocturnal results demonstrated that whereas Ad dropped during the supine resting, NA did
not fall until SWS period. Although DA did not show significant changes during the nocturnal
test, the NA/DA ratio showed significant reduction. The analysis of correlations supports the
postulation that this finding reflects the DA modulatory role on neural sympathetic activity.
Both f-5HT and p-5HT values were lower during sleep cycle than wake periods. However,
they showed progressive rises during sleep stages. Conversely, the f-5HT/p-5HT ratio showed
significantly greater values during the SWS period than during supine resting and REM per-

Corresponding author. Apartado. 80.983, Caracas 1080-A, Venezuela. Tel.: +58-212-961-1048; fax:
+58-212-961-0172.
E-mail address: flechin@telcel.net.ve (F. Lechin).
0306-4530/$ - see front matter 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0306-4530(03)00095-7

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F. Lechin et al. / Psychoneuroendocrinology 29 (2004) 669685

iods. These findings are consistent with the postulation that f-5HT/p-5HT ratio is positively
associated with parasympathetic activity during the sleep-cycle. We concluded that the profile
of sleep-cycle circulating neurotransmitters differs from that obtained during waking periods.
According to the above, we attempted to correlate the profile of circulating neurotransmitters
with the very well-known central neurocircuitry functioning during wakesleep cycle, in
experimental mammals.
2003 Elsevier Ltd. All rights reserved.
Keywords: Plasma catecholamines; Plasma serotonin; Platelet serotonin; Plasma tryptophan; Sleep; Wake
sleep cycle

1. Introduction
Circulating neurotransmitters: noradrenaline (NA), adrenaline (Ad), dopamine
(DA), free serotonin (f5HT), plasma tryptophan (TRP) and platelet serotonin (p5HT)
have been routinely investigated during the last 22 years in some 25,000 normal and
diseased subjects (Lechin and van der Dijs, 2002). These parameters have been
assessed during diurnal supine resting, orthostasis and moderate exercise conditions
(Lechin et al., 2002a). In addition, we also have tested the effects of oral glucose
as well as many central-acting drugs such as clonidine, buspirone, atropine, etc. on
these neurotransmitters (Lechin et al., 1985a, b, 1987; 1990a, b, 1992, 1993, 1995a,
b, 1996a, b, c, 1998). Other authors have also investigated plasma catecholamines
associated with the wakesleep cycle, however, they did not include plasma indoleamines (Prinz et al., 1979, 1984; Sowers and Vlachakis, 1984; Cameron et al., 1987;
Kuchel and Buu, 1985; Dodt et al., 1997); and in addition, these fragmentary studies
did not investigate the different portions of wakesleep cycle. Taking into account
that there exists poor knowledge dealing with the interactions between central neurocircuitry and peripheral neuroautonomic system, we decided to investigate all circulating neurotransmitters, not only during wake but also during sleep cycle, in order
to obtain some information about the central and peripheral interaction.
In the present study, we show results obtained in 20 normal subjects during 30
min of supine resting (sr-1), 1 min of orthostasis (ort) and 5 min of moderate exercise
(exc = moderate walking). This study was carried out in the morning. The same
subjects were investigated at night (the same day) during the sleep cycle, including
supine resting (sr-2), stage 2 sleep (S2), slow wave sleep (SWS) and rapid eye movement sleep (REM). The results obtained in the sleep cycle were correlated with those
obtained during the wake cycle, allowing us to postulate a possible parallelism
between the peripheral neuroautonomic profile and the very well-known central neurophysiological mechanisms underlying the sleep cycle.

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671

2. Methods
2.1. Subjects
Subjects were selected from a pool of undergraduate and graduate students after
an initial screening interview performed by a team of psychologists. Hamilton (1960)
(anxiety and depression) rating scales were routinely evaluated in all subjects. Subjects with sleep complaints, tobacco, alcohol or drug abuse and any medical or mental
illness were excluded. Initially, 28 healthy paid volunteers were chosen to participate
in this 3-day study, 13 women and 15 men, aged 1742 years. All were normotensive,
non-smoking students in good physical and mental health. Their body mass index
was between 2025 kg/m2. They took no psychoactive or neuroleptic drugs during
the eight weeks prior to the study. All were exhaustively evaluated (physically, endoscopically, radiologically, biochemically, bacteriologically, immunologically and
psychiatrically) in order to rule out any physical or mental illness able to modify
circulating neurotransmitters or other physiologic parameters. Finally, the 20 subjects
rated as good sleepers (according to the second night investigation) participated in
this study: 11 women and 9 men, ages 1733, age range 24.08 1.33 (mean
SE). Sleepers rated as good were those who after a normal sleep latency, showed
two or more complete no-REM (stages 1, 2, delta sleep)REM cycles followed by
several REM cycles in the second part of the night. Their average sleep efficiency
was 92%. Informed consent was obtained from all individuals. The experiments were
performed in the Sleep Research Lab of the Section of Neuropharmacology, Instituto
de Medicina Experimental, Universidad Central de Venezuela.
2.2. Polysomnographic evaluation
A full electrode standard sleep montage included EEG (C3, C4, O1, O2), chin
EMG (bipolar chincheek electromyograms), EOG (left and eye electrooculograms),
both tibial leg muscle electromyograms, EKG (bipolar electrocardiogram), chest and
abdomen leads, a nasaloral thermistor, a finger oxymeter, and a position sensor.
The EEG and EOG monopolar leads were referred to the opposite mastoid electrode.
Data acquisition, analysis and reports were performed on a computer-aided system
using REMBRANDT softwares (Data Lab and Report Manager) (Medcare,
Buffalo, NY, USA). Sleep scoring was visually done at 30-s intervals according to
Rechtschaffen and Kales (1968) criteria. Sleep onset was defined as the first 30-epoch
scored SWS-2. All the PSG automated-analyzed signals were graphically edited.
2.3. Experimental protocol
In the last day of this 3-day study, we measured levels of plasma NA, Ad, DA,
free serotonin (f-5HT), TRP and platelet serotonin (p-5HT) during the morning
supine resting, orthostasis and exercise test as well as during nocturnal supine resting
(drowsiness), S2, SWS and REM test in 20 healthy volunteers. Both tests were carried out during the same day. On the experimental 24 h period, after overnight fast-

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ing, subjects arrived at 07:00 h to have a supine resting, orthostasis, exercise test.
Blood samples were obtained by means of a heparinized venous catheter inserted
into a right forearm vein at least 30 min before beginning the test. All blood samples
were obtained between 07:00 and 09:00 h in order to control for diurnal variations,
in a temperature controlled room (6972 F) after the subject had been lying down
for 30 min with his or her right arm lying flat on a table and legs uncrossed. After
drawing the first sample (supine resting - 1 = sr - 1), the subject was instructed to
stand beside the examination bed, balanced on both feet and without leaning against
the bed. Thirty seconds after the subject assumed standing position the second blood
sample was taken (orthostasis = ort) and the subject was instructed to perform a
treadmill test consisting of 5 min exercise at 2 mph with the grade adjusted according
to subjects weight to give a total 6000 foot-pounds of work and then the third blood
sample was taken (exercise = exc).
Subjects were asked not to have a nap and refrain from consuming caffeine and
alcohol drinks during the day. They were also instructed to take an early supper
(18:3019:00 h) devoid of dessert, chocolate, cheese, snacks, beans and fried food
(a food diary was kept by subjects). They entered the sleep laboratory at 20:00 h.
They rested quietly for 2 h after which they went to bed. In a relaxing environment
the electrodes were attached. An indwelling polyethylene catheter was inserted in a
vein of the subjects arm so as to cause neither discomfort nor hindrance of movements during sleep. Those who showed some anxiety or discomfort were excluded.
The catheter was connected to a three-way luer lock in the next room by a long fine
plastic tube passing through a small hole in the wall. A slow-drip saline solution
(40 drops/min) was infused during the night by the catheter. Blood samples were
also taken from the adjacent room to avoid disturbing the subjects sleep. Subjects
rested supine for 15 or more min before a basal first blood sample was drawn.
Calibration procedures followed and lights were off about 22:30 h 6.2 min
(mean SE) when the sleep recording started. Blood was drawn after drainage of
saline solution from the catheter. This mixed blood plus saline solution was reinjected after the blood samples were drawn. Blood extraction was done under constant polysomnographic sleep monitoring and was aborted if an arousal was detected.
Blood samples were drawn after the first 3 min of stable S2 and 3 min of stable
SWS, as well as during the second REM sleep cycle. The sleep recording ended at
06:00 h. All subjects were submitted to two previous habituation sessions during
which the electrodes were attached and the catheter was inserted into the vein, but
neither blood samples nor sleep records were obtained. Blood tests and polysomnographic records were taken during the third night, only.
The investigation was performed according to the Declaration of Helsinki and was
approved by the Ethical Committee of FundaIME.
2.4. Analytical methods
Blood samples for measuring circulating neurotransmitters and tryptophan were
collected into plastic tubes containing an anti-oxidant solution (20 mg of EDTA plus
10 mg of sodium metabisulphite per ml). Platelet-rich plasma (PRP) was prepared

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673

by centrifuging the blood samples at 350 g at 4 C for 15 min and 2 ml of supernatant

were processed immediately for determination of p5HT levels. The remaining blood
was centrifuged again at 33,000g at 4 C and two aliquots of the supernatant, plateletfree plasma (PFP), were also immediately processed for determination of catecholamines, plasma free serotonin and tryptophan concentrations, respectively. A
10 l aliquot of PRP was used for counting platelets in a Platelet Counter model
MK-4/HC (Baker Instrument, Bethlehem, PA, USA). A 10 l aliquot of PFP was
also counted to ascertain the absence of platelet. The processed samples were stored
at !80 C until injected in the HPLC.
2.5. Analytical assays
Concentrations of catecholamines, serotonin and tryptophan were measured using
high-performance reverse-phase liquid chromatography with electrochemical detection (Davies and Molyneux, 1982; Eisenhofer et al., 1986; Kumar et al., 1990).
Optimization of chromatographic conditions allowed us maximal sensitivity and
reproducibility.
Catecholamine assays were performed by extraction onto 20 mg of alumina followed by their elution with 200 l of 0.2 mol/l HClO4, using BAS (Bioanalytical
Systems) microfilters. The instrument was previously calibrated with standard
plasma: after incubation with acid-washed aluminum oxide, a plasma pool free of
catecholamines was processed similarly to plasma samples, but 20 l of a standard
solution of NA, Ad and DA (50 ng/ml each) were added to 1 ml of the plasma pool.
Both standard plasma and sample plasma were supplemented with 20 l of internal
standard solution (dihydroxybenzylamine 100 ng/ml). The mobile phase was composed of KH2PO4 6.8045 gr/l, EDTA 0.100 gr/l, sodium octyl sulfate 0.55 gr/l, diN-bultylamine 100 /l, and acetonitrile 3.8% (v/v) with pH adjusted to 4.52. Concentrations are expressed in pg/ml. The intra-assay coefficients of variation were 2.5%,
3.7% and 3.8% for NA, Ad, and DA, respectively. The inter-assay coefficients of
variation were 5.5%, 3.9% and 4.1%, respectively.
Serotonin assay was performed after sonication of PRP to disrupt any intact platelets (Ultrasonic Liquid Processor, model 385, Heat Systems Ultrasonic, Inc., Farmingdale, NY, USA), both PRP and PFP were processed in the same way: 200 l of
3.4 M perchloric acid as deproteinizing agent and 10 l of 5-OH-tryptophan solution
(80 g/ml), as internal standard, were added to 1 ml of plasma, vortexed and centrifuged at 10,000 rpm 15 min at 4 C. The supernatant was filtered through a 0.2
m syringe filter and injected into the HPLC. The HPLC-ED system was previously
equilibrated and calibrated. Calibration runs were generated by spiking plasma blank
with 50 l of 5HT solution (10 g/ml) and 10 l of 5-OH-tryptophan solution (80
g/ml). This standard plasma was processed in the same manner as samples. Serotonin was determined after injection of 100 l of the deproteinized sample into a
Discovery column, 25 cm 4 mm i.d., packed with C18 5 m fitted to a 3 cm
precolumn filter 0.5 m. Mobile phase was composed of citric acid 3.8424 gr/l,
sodium acetate 4.1015 gr/l, EDTA 0.100 gr/l, octyl sulphate 0.015 gr/l, dibutylamine
25 l/l, propanol 4.3% v/v and pH adjusted to 4.0. It was delivered at a flow rate

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of 0.6 ml/min. The sensitivity of this method was 0.06 ng/ml; intra-assay coefficients
of variation were 4.1% for platelet-rich plasma serotonin and 4.4% for platelet-poor
plasma serotonin, respectively. Inter-assay coefficients of variation were 5.0% and
7.1%, respectively. Concentrations are expressed in ng/ml. Platelet serotonin value
= PRP serotonin value (total circulating serotonin)!PFP serotonin value (f5HT).
2.6. Platelet aggregation
Blood was collected with citrate-phosphate dextrose (1:9 v/v) as the anticoagulant.
Blood was subsequently centrifuged at 120g for 10 min to prepare PRP. Aggregation
studies were carried out according to Borns method (1962), and aggregation was
induced by ADP and collagen at final concentrations of 4 mol/l and 4 g/ml,
respectively. Maximal aggregation, expressed as the percentage of maximal light
transmission, was measured.
2.7. Statistical analyses
Results are expressed as mean SE. Multivariate ANOVA with repeated measurements, paired t-test, and Pearsons correlation coefficients (exploratory factor
analysis) were employed in interpreting the data yielded by this investigation. Differences were considered significant at p " 0.05. Only the variables of interest are
presented. SPSS program, version 9.0 for Windows, was used for graphics and statistical analyses.

3. Results
3.1. Wake cycle
NA showed significant increases at both orthostasis and exercise, p " 0.001 in
both cases. Ad showed significant increase at exercise but not at orthostasis (p =
n.s. and p " 0.001, respectively). DA did not show significant change at any period.
NA/Ad ratio showed significant and progressive increases at both orthostasis and
exercise periods (p " 0.05 and p " 0.001, respectively). NA/DA ratio showed significant and additional increases at both orthostasis and exercise periods (p "
0.02; p " 0.001, respectively). Ad/DA ratio showed significant increase at exercise,
only (p = n.s. and p " 0.005, respectively) (Table 1, Fig. 1).
f-5HT did not show significant oscillation at any period (p = n.s. at all periods).
p-5HT did not show significant change at any period (p = n.s. at all periods). f5HT/p-5HT ratio showed significant drop at orthostasis but not at exercise (p "
0.005 and p = n.s., respectively). TRP did not show significant change at any period
(p = n.s. at all periods) (Fig. 2).

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675

Table 1
Circulating neurotransmitters during diurnal wake cyclea

Noradrenaline
Adrenaline
Dopamine
NA/Ad
NA/DA
Ad/DA
f5HT
p5HT
Tryptophan
f-5HT/p-5HT
a

Units

Supine resting

Orthostasis

Exercise

pg/ml
pg/ml
pg/ml

73.5 7.07
21.9 2.40
33.8 16.1
3.12 0.37
3.46 1.0
1.45 0.24
5.27 2.69
464.8 49.84
8672 794.3
0.019 0.0035

102.2 15.5
25.38 3.68
25.4 3.68
4.5 0.32
5.98 1.07
1.56 0.29
4.57 1.52
474.9 40.09
8848 768.6
0.017 0.0038

172.9 25.0
33.1 4.20
20.3 5.10
5.7 0.76
11.3 2.76
2.36 0.43
5.43 1.83
469.7 47.79
8948 695.7
0.012 0.010

ng/ml
ng/ml
ng/ml

Values are given as mean SE.

3.2. Sleep cycle

Noradrenaline (NA) supine resting (sr-2) value showed significant and progressive
reduction during all sleep stages: S2, SWS and REM. NA levels reached minimal
value (near to zero) at REM period. A significant fall of Ad was registered at REM
stage when compared with sr-2. The NA/Ad ratio showed progressive and significant
reductions that paralleled the NA curve These results indicate that although prolonged supine resting state is shown to reduce adrenal sympathetic activity to almost
minimal level, neural sympathetic activity remains elevated while the subject is
awake.
Dopamine (DA) plasma levels showed non-significant changes during the nocturnal sr-2/S2/SWS/REM test. However, DA values tended to be higher during the SWS
period (Table 2).
Noradrenaline/dopamine (NA/DA) ratio showed a profile, which parallels the NA
and NA/Ad profile. However, significant and important differences were found with
respect to correlations registered between NA/DA values and NA/Ad absolute values
when plotted vs. other parameters.
Adrenaline/dopamine (Ad/DA) ratio showed values resembling the Ad profile.
However, Ad values but not Ad/DA values showed significant reduction during the
REM sleep stage.
Free serotonin (f-5HT) in the plasma showed values demonstrating non-significant
changes during the sr-2/S2/SWS/REM test. However, f-5HT tended to rise during
SWS stage. In addition, f-5HT values, which showed significantly lower values than
those found during morning periods, tended to fade at REM sleep stage.
Platelet serotonin (p-5HT) showed significantly lower levels than those registered
during diurnal periods. However, these values did not show significant changes during the sr-2/S2/SWS/REM nocturnal test.
Tryptophan (TRP) in the plasma did not show significant changes during the sr2/S2/SWS/REM test. However, TRP values tended to lower during REM stage and,

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F. Lechin et al. / Psychoneuroendocrinology 29 (2004) 669685

Fig. 1. Noradrenaline, adrenaline, dopamine, and NA/Ad plasma values (mean SE) obtained from 20
normal subjects during supine resting (sr-1)/orthostasis (ort)/exercise (exc) morning test and supine resting
(sr-2)/S2/SWS/REM nocturnal test. Both test were performed the same day. () () () = levels of
significance vs. sr-1 and sr-2, respectively.

in addition, the TRP plasma levels tended to rise during the late nocturnal periods
(not presented results) (Fig. 3).
Free serotonin/platelet serotonin (f-5HT/p-5HT) ratio showed significantly greater
values during SWS period than at sr-2 and REM periods. However, the f-5HT/p5HT ratio at SWS period was significantly lower than those registered at sr-1 and
ort diurnal periods.
4. Discussion
The results obtained from the present study showed that whereas Ad is significantly reduced during nocturnal supine resting state (sr-2), NA is not. NA plasma

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677

Fig. 2. Noradrenaline/dopamine (NA/DA) ratio, adrenaline (Ad)/DA (Ad/DA) ratio, and free serotonin
(f5HT/platelet serotonin (p5HT) (f5HT/p5HT) ratio values (mean SE) obtained from 20 normal subjects
during supine resting (sr-1)/orthostasis (ort)/exercise (exc) morning test and supine resting (sr2)/S2/SWS/REM nocturnal test. Both test were performed the same day. () () () = levels of
significance vs. sr-1 and sr-2, respectively.

levels showed progressive reduction during S2 and SWS and reached minimal levels
at REM sleep period. Although Ad did not show continued reduction during SWS,
a final significant fall was registered during REM sleep. These different profiles of
NA and Ad during supine resting-2 and sleep cycle indicate that the two catecholamines arise from two different sources: sympathetic nerves and adrenal glands, respectively. This phenomenon registered during sleep cycle was also evident during wake
cycle (short-lasting orthostasis), at which stage NA but not Ad was raised in the
plasma. These findings demonstrated that dissociation between neural and adrenal
sympathetic activities is a physiological phenomenon. However, both sympathetic
activities can function in association during the wakesleep cycle, for instance during
exercise, SWS and REM sleep. Nevertheless, the contribution of each component
of the sympathetic system would depend on the circumstances, either physiological

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Table 2
Circulating neurotransmitters during nocturnal sleep cyclea

Noradrenaline
Adrenaline
Dopamine
NA/Ad
NA/DA
Ad/DA
F5HT
P5HT
Tryptophan
f-5HT/p-5HT
a

Units
pg/ml
pg/ml
pg/ml

ng/ml
ng/ml
ng/ml

sr-2
79.6 23.3
19.3 2.02
15.5 2.52
3.88 0.65
5.2 0.77
1.41 0.16
2.19 0.53
290.7 30.9
8882 540
0.0054 0.009

S2
44.0 5.1
17.1 1.46
20.7 5.51
2.57 0.20
3.02 0.44
1.18 0.19
3.37 1.27
320.8 32.2
88468 471
0.0129 0.006

SWS
35.6 3.28
17.4 1.61
17.7 3.72
2.21 0.25
2.65 0.33
1.23 0.17
3.47 1.03
327.3 29.8
8286 571
0.0128 0.005

REM
23.3 2.34
15.2 1.79
14.9 1.7
1.68 0.19
1.8 0.24
1.13 0.15
2.35 0.58
341.1 30.9
7752 571
0.0072 0.002

Values are given as mean SE.

or pathophysiological. This issue has been discussed by us in many other published

papers (Lechin et al., 1993, 1995a, b, 1996a, b, c, 1998).
The association or dissociation of neural sympathetic vs. adrenal sympathetic
activity is consistent with the NA vs. Ad correlations registered during nocturnal
period. NA-sr-2 vs. NA-S2 showed highly significant positive correlation (p "
0.001). This significance disappeared when NA-sr-2 was plotted vs. NA-SWS and
NA-REM (p = n.s.).
NA-sr-2 showed significant correlation when plotted vs. Ad-sr-2 (p " 0.005);
however, no significant correlations were found when NA-sr-2 was plotted vs. AdS2, Ad-SWS and Ad-REM, respectively.
Although DA plasma levels did not show significant changes during the wake
sleep cycle, they tended to decrease (like NA and DA) during sr-2. DA showed
slight non-significant increases during SWS and REM periods. These findings are
consistent with the significant NA vs. DA positive correlation at sr-2 (p " 0.005),
but negative correlations were registered during S2 and SWS (p " 0.001 in both
cases).
Comparison of the NA/Ad ratio with the Ad/DA ratio merits some discussion.
The NA/DA ratio showed significant reduction during SWS, a period when NA
showed lower levels than those registered at S2, hence this NA/DA reduction at
SWS would be attributed to the significant NA decrease plus the non-significant DA
increase. This DA increase occurred during a period when adrenal glands secretion
reached minimal level, as revealed by the Ad and Ad/DA ratio values. Thus, it is
reasonable to assume that the minimal release of NA from sympathetic nerves is
related to an increase of DA from the latter. These facts fit clearly with the wellknown inhibitory role exerted by the DA pool existing at sympathetic nerves
(Wilffert et al., 1984; Soares Da Silva, 1986, 1987). Summarizing the above, the
supine resting state inhibits adrenal gland secretion but not sympathetic nerve release
of NA + DA, when progressive reduction of NA but not DA release from sympathetic
nerves is produced. These findings are consistent with the fact that the Locus Coeru-

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679

Fig. 3. Free serotonin, platelet serotonin and tryptophan plasma values (mean SE) obtained from 20
normal subjects during supine resting (sr-1)/orthostasis (ort)/exercise (exc) morning test and supine resting
(sr-2)/S2/SWS/REM nocturnal test. Both test were performed the same day.

leus NA pontine nucleus shows progressive fading during SWS and its NA neurons
are silent during REM sleep (Trulson and Jacobs, 1979, 1981; Lechin et al., 2002b).
Both free serotonin (f-5HT) and platelet serotonin (p-5HT) showed significantly
lower values during the sleep cycle than those registered during the wake cycle.
Tryptophan levels, although showing no significant differences between wake and
sleep cycle, tended to lower at the end of the sleep cycle. These findings are consistent with the well-known fading of central serotonergic activity during sleep.
The free serotonin/platelet serotonin (f-5HT/p-5HT) ratio showed two minimal
levels, at sr-2 and REM sleep periods, which differed significantly from the two
maximal values registered at S2 and SWS period. These findings would be associated
with small increases of DA at the two last periods, which interfere with the platelet
uptake of serotonin.
In the absence of increased platelet aggregation (Haft and Arkel, 1976; Brammer
et al., 1982; Levine et al., 1985; Larsson et al., 1989; Karege et al., 1993), (all our

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F. Lechin et al. / Psychoneuroendocrinology 29 (2004) 669685

subjects showed non-increased platelet aggregability), the values of the f-5HT/p-5HT

ratio would depend on the uptake of 5HT by platelets, mainly. This uptake is interfered by acetylcholine (ACh) (Rausch et al., 1985) and DA (Kerry and Scrutton,
1985; Ricci et al., 2001). The rises of the f-5HT/p-5HT ratio demonstrated during
S2 and SWS are consistent with both the hyper-parasympathetic activity known to
exist at this period (Maling et al., 1979) and the relative DA rise (with respect to
NA and Ad) also demonstrated in our study.
It is not possible to assess plasma ACh. This neurotransmitter is rapidly hydrolyzed
by acetylcholinesterase existing in the plasma. However, hyper-parasympathetic
activity during sleep has been shown to exist by many direct and indirect physiological parameters (Maling et al., 1979). Thus, it is accepted that during this period
parasympathetic nerves release ACh, which accedes to the bloodstream. According
to all the above, it is logical to assume that the f-5HT/p-5HT ratio would increase
during sleep and other hyper-parasympathetic situations. In this regard, it has been
shown that all increases of plasma f-5HT not associated with increased platelet
aggregability are accompanied by blood pressure and heart rate falls. These have
been suppressed and/or prevented by atropine (0.5 mg, intramuscularly injected)
(Lechin et al., 1996b; Lechin, 2000). Finally, maximal reductions of both NA and
Ad, but not DA, registered during SWS are consistent with the accepted knowledge
that maximal parasympathetic activity occurs at this period. According to all the
above, we postulate that the f-5HT/p-5HT ratio is a trustworthy index of parasympathetic activity during physiological and some pathophysiological conditions.
4.1. Nocturnal indoleamines vs. nocturnal indoleamines
Free serotonin (f-5HT)-sr-2 values did not correlate when plotted vs. f-5HT-S2
and f-5HT-SWS, periods when small but non-significant f-5HT rises were registered.
However, highly positive correlation was found between f-5HT-sr-2 vs. f-5HT-REM,
at which period f-5HT dropped (p " 0.005). In addition, the fact that both f-5HTS2 and f-5HT-SWS showed highly positive correlations when plotted vs. f-5HT/p5HT ratio, at both S2 and SWS period (p " 0.000 in both cases) supports our
postulation that the f-5HT/p-5HT ratio reflects parasympathetic activity as maximal
parasympathetic activity (acetylcholine = ACh plasma levels) is registered in phases
S2 and SWS of the sleep cycle.
4.2. Nocturnal catecholamines vs. nocturnal indoleamines
The most distinctive finding yielded by the analysis of these results is the significant positive correlation between NA vs. p-5HT. This finding is consistent with the
demonstrated fact that both parameters fell during nocturnal periods. The p-5HT fall
occurred during sr-2 period and remained lowered during all sleep stages. NA fell
later than p-5HT, at S2. For this reason, the NA vs. p-5HT significant positive correlation was registered at SWS (p " 0.005), but not at sr-2 period nor at REM period
(p = n.s.).
The fact that NA and NA/Ad but not DA plasma values showed significant and

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681

progressive reductions during SWS and REM periodsreductions that were paralleled by a central NA and DA dissociated profile of neural activityshould be discussed in the light of findings arising from this study. It should be remembered that
whereas NA neurons (locus coeruleus) show progressive reduction of activity during
SWS and REM periods (Aston-Jones and Bloom, 1981; Lechin and van der Dijs,
1984; Gaillard, 1985; Aston-Jones et al., 1991; Haddjeri et al., 1997), DA neurons
(A8, A9) maintain a profile of activity during all sleep stages, while A10 mesolimbic
(subcortical) group shows a reduction of activity during all sleep phases (Lechin and
van der Dijs, 1984). Knowledge of anatomical plus functional interactions aids in
the understanding of neurotransmitter interactions. It should be remembered that subcortical DA neurons receive inhibitory inputs from NA neurons (LC) whereas mesocortical DA neurons receive excitatory LC-NA axons (Anden and Stro mbom, 1974;
Anden and Grabowska, 1976; Kostowski, 1979; Grenhoff and Svensson, 1993). In
addition, subcortical DA neurons receive excitatory axons from the midbrain ACh
neurons (Grillner et al., 2000) including the pedunculo-pontine nucleus (Reese et al.,
1995). Furthermore, LC-noradrenergic neurons show increased activity during wake
cycle (McGinty and Harper, 1976; Lechin and van der Dijs, 1984; Gaillard, 1985;
Aston-Jones et al., 1991), whereas ACh neurons are highly active during sleep periods (maximal activity at REM sleep) (McGinty and Harper, 1976; MacCarley, 1982;
Lechin and van der Dijs, 1984; Dube et al., 1985; Lechin et al., 1992, 2002a, b).
Thus, DA mesocortical neurons are stimulated by NA axons during the active waking
periods and subcortical DA neurons are activated by ACh axons during sleep stages.
NA released from NA axons acts on excitatory alpha1 receptors (Grenhoff and Svensson, 1993), whereas ACh axons act on muscarinic receptors. This NADAACh
central circuitry is paralleled at peripheral level by the two components of neural
sympathetic activity (NA and DA) plus parasympathetic activity (ACh). Finally,
adrenal sympathetic activity (adrenal glands) is ruled by the Ad-C1 medullary nuclei
(located at the lateral rostral area). Central and peripheral adrenal sympathetic activities remain silent during sr-2/S2/SWS/REM cycle.
4.3. Diurnal catecholamines vs. nocturnal indoleamines
Neural sympathetic activity (NA/Ad) at orthostasis (ort) showed high positive correlation vs. nocturnal p-5HT; p " 0.001, p " 0.002, and p " 0.02 when plotted vs.
sr-2, S2 and SWS period, respectively. Considering that adrenal glands (adrenal sympathetic activity) do not increase Ad secretion at orthostasis, the above mentioned
positive correlation indicates that the greater the diurnal neural sympathetic activity,
the greater the nocturnal levels of p-5HT. These NA/Ad-ort vs. nocturnal p-5HT
positive correlations are also registered during the diurnal supine resting (sr-1), and
exercise (exc) periods but not during the nocturnal supine resting (sr-2), stage 2 (S2),
SWS and rapid eye movement (REM) stages. Thus, the positive NA vs. p-5HT
correlation is not present during sleep periods.

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F. Lechin et al. / Psychoneuroendocrinology 29 (2004) 669685

4.4. Nocturnal catecholamines vs. diurnal indoleamines

The negative correlation registered for diurnal TRP vs. nocturnal Ad-sr-2 (p "
0.005), as well as the positive correlation of the former vs. nocturnal NA/Ad ratio
(p " 0.005) are consistent with the well-known NA vs. 5HT central antagonism.
On the other hand, the TRP vs. Ad antagonism reinforces the postulation that plasma
TRP is positively correlated with peripheral parasympathetic activity. The above
postulation is reinforced by the close positive correlation found between diurnal f5HT vs. nocturnal f-5HT and f-5HT/p-5HT values (p " 0.005 and p " 0.002,
respectively).
4.5. Nocturnal indoleamines vs. diurnal indoleamines
Nocturnal values of f-5HT at supine resting and REM periods showed highly
significant correlations vs. diurnal f-5HT at supine resting (p " 0.001) and orthostasis
(p " 0.001) periods but not at exercise period, at which time both plasma NA and
Ad are raised. These findings ratify results shown in other tables which support the
postulation that f-5HT level depends on two opposite factors: (a) platelet uptake,
and (b) platelet release of 5HT. Platelet uptake is interfered by ACh and DA, whereas
platelet release is triggered by plasma Ad, which favors platelet aggregation.
Similar positive correlations were registered between f-5HT-sr-2 and f-5HT/p-5HT
ratio at sr-1 and orthostasis (p = 0.000, and p " 0.001, respectively), but not during
exercise. These findings are consistent with others showing that f-5HT/p-5HT ratio
is positively associated with parasympathetic activity (plasma ACh).
Nocturnal values of tryptophan (sr-2, S2, SWS and REM) showed highly positive
significant correlations when plotted vs. TRP-sr-1 (p " 0.005, p " 0.002, p "
0.005 and p " 0.002, respectively) and TRP-ort (the latter lower than the former)
(p = n.s., p " 0.005, p " 0.005, and p " 0.005, respectively). Conversely, no
correlation was found between TRP-sr-2 vs. TRP-exc when maximal levels of catecholamines in the plasma were registered.
Summarizing, the global analysis of correlations presented in this study allows us
to support the statements below.
Nocturnal neural sympathetic activity remains high during sr-2 and S2 but falls
at SWS and REM periods, whereas nocturnal adrenal sympathetic activity suffers
deep reduction as of sr-2 period. Both sympathetic activities remain constant during
SWS but register additional fall at REM period. According to the above, neural and
adrenal sympathetic activities function dissociatedly during sr-2 and S2, but are in
parallel during SWS and REM periods. Further, not only diurnal but also nocturnal
DA plasma levels arise from sympathetic nerves rather than adrenal glands. The NA
vs. DA negative correlations registered during SWS and REM would be related to
the inhibitory role exerted by the DA pool existing at sympathetic nerves.

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683

5. Acknowledgments
This work was supported by grants from FundaIME and FundaNEUROINMUNOLOGIA. We thank Arnoldo Rangel, MSc, for his technological assistance in polysomnography in the completion of this research.

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