1. Neuroscience Vol. 79, No. 3, pp. 683–693, 1997
Copyright 1997 IBRO. Published by Elsevier Science Ltd
Printed in Great Britain. All rights reserved
Pergamon PII: S0306-4522(96)00709-9 0306–4522/97 $17.00+0.00
DETERMINATION OF NADH IN THE RAT BRAIN DURING
SLEEP-WAKE STATES WITH AN OPTIC FIBRE SENSOR
AND TIME-RESOLVED FLUORESCENCE PROCEDURES
S. MOTTIN,* P. LAPORTE,* M. JOUVET† and R. CESPUGLIO†‡
*T. S. I. Laboratory, CNRS-URA842, Jean Monnet University, F-42023, St-Etienne, Cedex 02, France
†Department of Experimental Medicine, INSERM-U52 CNRS-ERS5645, F-69373 Lyon,
Cedex 08, France
Abstract––The present paper reports a nanosecond time-resolved fluorescence derived from the cortex and
the area of the periaqueductal gray including the nucleus raphe dorsalis (PAG-nRD) in unanaesthetized
freely moving rats. The measurements were acquired through a single optic fibre transmitting a
subnanosecond nitrogen laser pulse (337 nm, 15 Hz) and collecting the brain fluorescence occurring at
460 nm which might depend on mitochondrial NADH (reduced form of nicotinamide adenine dinucle-
otide). The fluorometric method was combined with polygraphic recordings, and this procedure allowed
us to define, for the first time, variations of the 460 nm signal occurring throughout the sleep-wake cycle.
In the PAG-nRD, the signal exhibited moderate heterogeneous variation in amplitude during slow-wave
as compared to the waking state. Constant increases were observed during paradoxical sleep as compared
to the waking state. For this state of sleep the magnitude of the variations depended on the optic fibre
location. In the cortex and during either slow-wave sleep or paradoxical sleep, the signal presented
moderate increases which were significant during paradoxical sleep.
The magnitude of the redox variations observed either in the PAG-nRD or in the cortex might be
ascribed to the oxidative energy balance which is related to sleep states. 1997 IBRO. Published by
Elsevier Science Ltd.
Key words: optic sensor, laser, NADH, brain, sleep, rat.
The use of biochemical sensors or of permeation sensor (FOCS), easily implantable in the brain of
methods allowing measurement of brain-specific animals under chronic conditions and allowing fast
compounds is actually a well established procedure. measurements of the brain chemical contents in com-
These tools are suitable for behavioural studies since bination with sleep polygraphic recordings. Since this
they are mini-invasive and sufficiently sensitive to approach is applicable to several fields, for clarity’s
detect, for example, extracellular changes occurring sake we shall review the issues associated with each
in neurochemicals across sleep-wake alternations.9,74 field.
Biochemical sensors, through an electrochemical
detection, allow quasi-continuous measurements
Technical aspect
without causing significant disturbances to the brain.
Nevertheless, they are only suitable for a limited The first detailed spectrofluorometric study per-
number of compounds, generally the amines and formed in the brain used the ‘‘surface microfluorom-
their metabolites74, nitric oxide11,47 and glucose.72 etry’’ technique for detection of brain NADH
Permeation methods allow measurement of a greater fluorescence.12 Since then, the authors have also used
variety of substances, but are limited by their a Y-shaped optic fibre ( =200 µm) to monitor the
anatomical resolution and the time-lag necessary NADH fluorescence in the surface of the rat brain.50
to collect the fractions between successive These approaches have allowed the emergence of
measurements.4 multiple technical improvements, i.e. laser-induced
To overcome these difficulties, we introduced an fluorescence system, micro-optical fibre, fluorescence
alternative technique using a fibre optic chemical microscopy, video imagery and time-resolved fluor-
escence techniques.22,32,40,50,66,70 Lastly, and in ad-
‡To whom correspondence should be addressed. dition to the above aspects, light reflectance has also
Abbreviations: AcCoA, acetyl coenzyme A; ACh, acetyl- been measured from the dorsal hippocampus to
choline; CSF, cerebrospinal fluid; EEG, electroencepha- assess correlations with electroencephalographic
logram; EMG, electromyogram; FOCS, fibre optic activity.61
chemical sensor; 5-HT, serotonin; nRD, nucleus raphe
dorsalis; PAG, periaqueductal gray; PDH, pyruvate de- The autofluorescence measured by spectroscopic
hydrogenase; PS, paradoxical sleep; SWS, slow-wave methods is now believed to be dependent on the
sleep; W, waking state. mitochondrial NADH fraction contained within the
683

2. 684 S. Mottin et al.
volume probed 12,22,34,50 for the following reasons: (i) energy supply and, according to the oxidative phos-
NADH absorbs only in its reduced form at 340 nm phorylation processes, is first metabolized within the
and fluoresces within the range of blue light at glial cells into pyruvate which is then transported
440–530 nm;12 (ii) in the gray matter78 or in the into neurons.1,67 GLUT3 (glucose transporter, iso-
cortex,34 increased levels of the 450–460 nm autofluo- form 3) is believed to be the major neuronal glucose
rescence are well correlated with increased tissue transporter.28 At this level, mainly through the pyru-
levels of NADH and the decreased tissue contents in vate dehydrogenase complex (PDH), pyruvate is the
glucose and ATP;56 (iii) results reported by imaging source for the production of acetyl coenzyme A
studies in the surface of the brain also indicate that (AcCoA) and NADH:
the blue fluorescence is correlated with the distribu- Pyruvate+CoASH+NAD+ PDHAcCoA+
tion of the mitochondrial NADH fluorescence;22 (iv) NADH+H++CO2
finally, injection of cyanide (cytochrome oxidase Through allosteric regulations, PDH is inhibited
inhibitor) increases blue fluorescence while the injec- by the end products and activated by the substrates,
tion of FCCP (carbonylcyanide-p-trifluoromethoxy i.e. oxygen, Ca2+, pyruvate...57 AcCoA is the basic
phenylhydrazone), which uncouples phosphorylation fuel for the tricarboxylic acid cycle which produces
and oxidative processes, decreases it.22 NADH. Together with oxygen, and through
It must also be emphasized that changes occurring chemiosmotically-coupled oxidative phosphoryla-
in ‘‘NADH’’ fluorescence are complicated by the fact tion, it is highly ATP productive.23 It contributes to
that sampling performed with optic fibre sensors the synthesis of various chemical species like the fatty
involves the entire tissue sample. Indeed, assignment acids, the ketonic compounds, acetylcholine
of the compartment from which the signal arises (ACh)....29 NADH is reoxidized into NAD+ through
still remains to be precisely determined, and this redox processes of the proton motive respiratory
aspect has given rise to controversies.19,22,50 chain.6,53 It is thus evident that the above aspects of
Another essential issue concerns the conditions of oxidative metabolism depend on, and are reflected
the animals in reported experimental protocols. It is, by, the redox potential of the cells31 and that NADH
indeed, suggested19 that the ‘‘prestimulatory’’ steady may reflect the status of this aspect.3
redox state is of paramount importance in determin-
ing the direction of NAD/NADH redox balance
Sleep and energetic aspects
evoked by activation of the brain cortex. Whether the
animals are habituated or not to experimental con- As yet, the brain energetic mechanisms involved in
ditions may lead to decreased or increased in brain sleep have not been extensively investigated. It is
contents of lactate and pyruvate.62 It is also likely nevertheless reported that during slow-wave sleep
that data obtained with anaesthetized animals may (SWS), glucose49 and oxygen consumption45,49 de-
not exactly reflect the genuine changes occurring in crease while glycogen content increases,37 thus con-
true physiological conditions. tributing to the restoration of the energy pool.30,37
Indeed, SWS is generally assumed to be a restorative
state5,38 necessary for the occurrence of PS77 which
Sleep aspect
needs energy to occur. In this respect, on the basis of
A great variety of data obtained a few decades deoxyglucose (614C) use through autoradiographic
ago35 or more recently10 still point to the leading role technique or positron emission tomography, in-
played by serotonin (5-HT) in sleep. This amine is creases in glucose consumption have been reported
synthesized within serotoninergic neurons whose during PS as compared to the waking state
perikarya are located in the raphe system and con- (W).25,44,48,60
tribute, together with hypnogenic substances, to sleep
preparation, triggering and maintenance.20,21 In ad-
General purpose
dition to the aspects relative to neurotransmission or
neuromodulation associated with 5-HT or hypno- Our experiments were conducted with animals
genic substances, the energetic mechanisms involved under strictly controlled physiological conditions.
in this critical area throughout the sleep-wake cycle Despite the evidence stated above, we first reconsid-
and particularly during paradoxical sleep (PS), re- ered the NADH dependence of the in vivo 460 nm
main poorly documented. The brevity of the PS signal since this aspect is essential for a sound in-
episodes displayed by most experimental animals terpretation of the data. Afterwards, we examined
could explain this issue. the relationship existing between the oxidative brain
metabolism and the vigilance states. For this pur-
pose, we investigated, throughout the sleep-wake
Energetic aspect
cycle, variations of the 460 nm fluorescence occurring
Energy is generally trapped as ATP by two pro- in the PAG-nRD, an area known to contain a large
cesses, glycolysis and mitochondrial oxidative phos- proportion of serotoninergic neurons79 as well as the
phorylation, whose respective efficiencies are 6% and PDH complex.52,57 Finally, we also investigated the
94%.23 In the rat, glucose is the basic substrate for frontal cortex where the axonal processes arising

3. Brain NADH determination with an optic fibre sensor 685
Fig. 2. General configuration of the experimental set-up
displayed for the experiments performed with unanaesthe-
tized and freely moving rats. The experimental sessions were
scheduled during the light period (12 h). F, filter; M, dich-
roic mirror; M1 mirror; L, lens; PMT, photomultiplier; PD,
photodiode.
Fig. 1. Schematic diagram of the optic fibre and the
polygraphic electrodes assembly. After surgery, 10 days
were necessary for recovery. Constant care was devoted to emerging from the inner edge by about 2 mm. Afterwards,
the animals, the cannula guide was also removed and the brain was removed, coronal sections (depth: 20 µm)
replaced every day. performed with a cryostat and classical Cresyl Violet
staining of brain cuts applied.
from the PAG-nRD area impinge. To our knowl- Data scoring and statistics
edge, the time-resolved measurement of brain fluor-
Polygraphic data were scored visually as previously de-
escence in deep tissue like the PAG-nRD area and its scribed20 while the fluorescent signals measured were stored
variations throughout the sleep-wake cycle have in a microcomputer and analysed by means of a home-made
never been reported. program. For statistics, an ANOVA followed by a multiple
range test (Bonferroni–Dunnet) were used (Fig. 5).
EXPERIMENTAL PROCEDURES Time-resolved fluorescence measurements
The experimental set-up used (Fig. 2) has been described
In vivo experimental procedure
elsewhere.54 Here, we recall briefly that the laser source used
OFA male rats (IFFA CREDO, n=5) weighing 260–300 g was a nitrogen laser delivering pulses of 300 ps (FWHM).
were anaesthetized with chloral hydrate (400 mg/kg, i.p.). The R3810 photomultiplier type (Hamamatsu) was selected
Two guide cannulas were then implanted in the PAG-nRD in gain in order to detect single events. The 50 output of
and the cortex58 (Fig. 1). The insertion was performed the photomultiplier was coupled to a transient digitizer
according to two different angulations (cortex: 45 /frontal (Tektronix Model 7912 AD mainframe, Model 7A19 ampli-
plane; nRD: 30 /sagittal plane). The animals were also fier unit, Model 7B90P time base unit). The fluorescence
equipped with electrodes necessary for polygraphic record- signal was averaged during 6 s with a laser repetition rate at
ings (electroencephalogram (EEG) and electromyogram 15 Hz. The fibre used was a step index multimode, model
(EMG): recording of the electrical activity of the cortex and PCS 200 (Quartz et Silice, core=200 µm). The measure-
of the neck muscles, respectively). Surgery, electrode produc- ments were volumetric since performed either in the extra-
tion and placement were performed as already described.20 cellular space or in the intracellular compartment.
After ten days of recovery (12 h:12 h light:dark, tempera- According to data already published12 and established by
ture at 24 0.5 C, food and water ad libitum), the FOCS means of an excitation wavelength at 337 nm applied on
was placed in the cannula from which it emerged by 2 mm brain slices, it appeared that the maximal volume probed by
(inner edge). Time-resolved fluorescence measurements, to- our sensor (diameter 200 µm, excitation at 337 nm) was
gether with polygraphic recordings, started immediately and about 0.063 µl. This contrasts with electrochemical methods
daily sessions (about 6 h) were possible for more than one detecting current at the active surface of the sensor and
month. At the end of each experimental session, the optical permeation methods using a membrane as active surface.
fibre was removed from the cannula and washed with milliQ
water. In vitro, a 15 µM NADH (Sigma) solution at pH 7
(phosphate buffer, Merck) was used as reference before and RESULTS
after in vivo sessions. To avoid cerebrospinal fluid overflow
between each experimental session, a metallic guide was Characteristics of the in vitro and in vivo signals
inserted into the cannula from which it emerged by 2 mm.
In vitro. A 15 µM NADH solution (pH 7,
Recording sites location temperature=24 C) yielded a typical fluorescence at
At the end of the different experimental sessions, the 460 nm (Fig. 3A). With the oxidized form of NADH
animals were killed with a lethal dose of barbiturates (NAD+) no fluorescent emission was obtained.
(Nembutal), recordings being continued during this step.
After death, the position of the working sensor was checked
in every animal by applying an anodic current (1 mA/2 s) In vivo. The signals derived from the cortex and
through an iron electrode inserted into the cannula and also the PAG-nRD area appeared in the spectral range

4. 686 S. Mottin et al.
Fig. 4. Upper part; typical PAG-nRD measurements (fluor-
escence at 460 nm) obtained throughout the sleep-wake
cycle. Data obtained are expressed with reference to the
waking state (100%). For each measurement the fluor-
escence pulses are integrated during 6 s (digitizer full scale at
20 ns). Lower part; hypnogram indicating the vigilance
states during which the fluorescence is measured. Note the
PS-related increases in fluorescence intensity. The small dots
refer to the measured values; the solid line is the best fit. line
Abscissae, time in minutes; Ordinates AU, arbitrary units.
For other abbreviations see also Fig. 1 and Fig. 3.
Fig. 3. A) Typical fluorescence spectra derived through the conditions, i.e. about 10 days after surgery. At the
optic fiber located in; the cortex (P) or the PAG-nRD area beginning, just after the first brain insertion of the
( ) during a single in vivo experimental session; a buffer FOCS, the signal obtained exhibited an intensity level
solution at pH 7 (filled line on grey columns, 1 column=1 which decreases exponentially (typical factor of 1.8)
experimental point); a NADH solution (O, 15 µm, pH 7).
Bars represent S.E.M. obtained with five measurements. It during about 90 min. Afterwards, the signal height
can be noticed that the signals derived from the cortex and increased slightly but continuously for 6–7 hours.
the PAG-nRD area, appear in the spectral range peaking After three to five insertions, performed during suc-
around 430–450 nm while, in vitro, the signal derived from a cessive sessions, these phenomena lessened, leading
NADH solution appears at about 460 nm; the shift ob-
to the occurrence of a signal that decreases just after
served in vivo is due to the presence of an unknown
compound peaking around 400 nm. It also appears that the the probe insertion for about 30 min and remained,
fluorescence level derived from the nRD is higher than that thereafter, stable as checked on special sessions ex-
derived from the cortex. Cx, cortex; NADH, nicotinamide tending up to 24 h. The magnitude of the ‘‘insertion
adenine dinucleotide, reduced form). B) Comparison be- effect’’ depends on the probe location and the
tween the typical fluorescence pulses obtained at 460 nm
from the two areas investigated (Cx and PAG-nRD) and mechanical stimulation that is inevitably applied
from a NADH solution (15 µM, pH 7) at 24 C. It can be during the brain insertion. The background noise,
noticed that decay times measured in vivo are longer than in attached to the signals measured in chronic condi-
vitro. Wavelength is expressed in nanometers (nm), time is tions, is mainly due to the laser impulse fluctuations.
expressed in nanoseconds (ns) and intensity in arbitrary
No noticeable changes occurred in the signal when
units (AU). For other abbreviations see Fig. 1 and Fig. 3A.
stretching or rolling-up of the optical fibre was
produced by the spontaneous movements of the
peaking around 430–450 nm (Fig. 3A). Their decay animal. Such a result is, at least partly, due to the
times were longer than in vitro (Fig. 3). Moreover, the fibre selected which is weakly sensitive to torsions
fluorescence level derived from the PAG-nRD area and rotations. Moreover, during experimental ses-
was constantly higher than that obtained in the sions the animal was on permanent visual control
cortex and the magnitude of the ratio PAG-nRD/ and its home cage was standing on a rotatable plate
cortex was 2 (Fig. 3). Finally, in the fluorescence which was manually moved to compensate torsions.
range 390–430 nm, either in the PAG-nRD or in the Such manual compensation was rather unfrequent,
cortex, an additional signal was present (Fig. 3). i.e. typically once per hour. To further limit spon-
According to our in vitro measurements, it might taneous movements, the fibre was also holded by a
depend on different endogenous fluorophores and a rubber wire attached to the ceiling of the home cage.
contribution of 5-hydroxyindole compounds is Data obtained have been expressed with reference to
possible.54 the waking state (100%, Fig. 4), since the changes in
the signal intensity occurring throughout the sleep-
Combined polygraphic and time-resolved fluorescence wake cycle were relative from one state to another.
measurements
Sleep-related changes of the fluorescence in the
Effect of the optic fibre brain insertion. Combined PAG-nRD. In this area, the intensity of the 460 nm
measurements were performed in long-term chronic fluorescence varied according to the vigilance state

5. Brain NADH determination with an optic fibre sensor 687
and the location site of the probe (Fig. 5). In its from the PAG-nRD area or the cortex, throughout
antero-medio-dorsal part (Fig. 5C), a slight decrease the rat sleep-wake cycle. Before discussing the signifi-
was measured during SWS/W (/:versus) while during cance of the observed phenomena, it is necessary to
PS/W a significant increase occurs. In its antero- carefully analyse the specificity of the measurements
dorso-lateral part (Fig. 5D), increases in the signal performed towards NADH. In this respect, data
height were observed either during SWS or PS/W. In from the literature, also obtained by means of
its ventrolateral part (Fig. 5B), the signal decreased spectroscopic methods in the brain,12,32,50 are in
significantly during SWS/W and during PS, while keeping with the results reported here. Indeed, in
decreasing/W, it increases/SWS. Laterally to the vivo, an excitation at 337 nm leads to the appearance
aqueduct, in a site not considered to be part of the of a fluorescent signal analogous to that observed in
PAG-nRD (Fig. 5A), very mild changes occurred vitro in the spectral window 460–520 nm with a
during either SWS or PS/W. In this site, the fluor- 15 µM NADH solution. The decay times measured in
escence level increase was significant during PS/W both conditions appear, however, to be slightly dif-
(Fig. 5A). Finally, outside of the PAG-nRD area and ferent and this discrepancy might be attached to the
in a mediolateral position, the signal remained stable fact that, in vivo, NADH exists either in a free form
during W, SWS and PS. or combined with enzymatic proteins. It is, indeed,
reported that the quantum efficiency as well as the
Sleep-related changes of the fluorescence in the mean lifetime of the protein-bound NADH is largely
cortex. Contrary to what was observed in the PAG- increased as compared to that of the free
nRD area, in the cortex, the fluorometric signal NADH.12,27,41,63 Moreover, the latter form also
exhibited homogenous variations, i.e. mild increases presents a biexponential lifetime resulting from the
during SWS or PS as compared to W. The variation complex photophysical properties of nicotinamide
of the signal measured during PS was, however, and the equilibrium existing at short distance be-
significantly different as compared to W. tween open/folded conformations of adenine and
1,4-dihydronicotinamide.16
Variations in the 460 nm fluorescence after a lethal However, NAD(P)H, also present in vivo, yields
dose of barbiturates. 1–2 min after injection of bar- a signal at 460 nm which, at first sight, might
biturates, the animal stopped breathing. Shortly after contribute to our 460 nm signal. It is, nevertheless,
and precisely when the heart beats become irregular, very unlikely since: (i) NAD(P)H-NAD(P) is
the signal height rose actively for 10 min and then present in much lower concentrations in the tissue
remained stable for another 30 min (Fig. 6). In the than NADH-NAD+;45 (ii) the fluorescence derived
cortex and the PAG-nRD, the anoxic/normoxic ratio from the cortex is well correlated with the NADH
of the 460 nm fluorescence was in the 1.6–2.4 range. endogenous concentrations,34 and (iii) the localized
It took about 2.6 min for the signal height increase to mitochondrial blue autofluorescence is altered by
occur in the cortex and 4.3 min in the PAG-nRD (tm, specific inhibitors of the oxidative metabolism.22
fitting function: 1-exp[ t/tm]). Finally, a perfect control, allowing one to check the
strict dependence of the 460 nm signal on the
DISCUSSION NADH tissue content, would consist in inhibiting
The present approach has been technically feasible its synthesis pharmacologically. Such a procedure,
on the basis of three major technical developments: currently used by bioelectrochemists to identify the
(i) delivery and collection of light signals (laser and chemical species in vivo9 is unfortunately not poss-
fluorescence) through a thin single optic fibre allow- ible with NADH since the substances altering its
ing fast measurements (10 s) together with a good production would immediately be lethal for the
anatomical resolution ( =200 µm); (ii) implantation animals. Whatever the difficulties, data reported
of a guide cannula into specific brain areas allowing here or available in the literature, support the
the insertion of the optical fibre in the freely moving hypothesis that protein-bound NADH might be the
animal for five to six weeks; (iii) simultaneous data compound that mainly contributes to the 460 nm
processing of the optic and polygraphic signals signal measured in vivo.
derived from the brain.
Particular care was constantly devoted to the ani- Compartment probed
mals. Only those in perfect condition were included
in the protocol. Moreover, the small diameter of the In the brain there are 3 main compartments, i.e.
optic fibre core used enabled us to check the changes vascular, intra- and extracellular. From the indica-
occurring in the signal height in limited areas of the tions mentioned in the present report, it seems
brain. likely that when the probe is inserted into the brain,
the measurements assess a maximum spherical vol-
ume of about 0.063 µl and that, in a first instance,
Measurement specificity
all three compartments might be involved. Regard-
The present work reports the changes occurring in ing the vascular compartment, the limited loss in
the intensity of the 460 nm emission, derived either sensitivity due to blood and observed with

6. 688 S. Mottin et al.
PAG-nRD area W SWS PS
A 100 0.26 (n=599) 100.5 0.2 (n=1029) 101.2 0.3 (n=402)
B 100 0.35 (n=1020) 99.1 0.47 (n=267) 102 0.59 (n=112)
C 100 0.45 (n=224) 99.1 0.47 (n=267) 102 0.59 (n=112)
D 100 0.18 (n=2896) 104.6 0.21 (n=1879) 106.6 0.46 (n=469)
E 100 0.3 (n=606) 100.7 0.26 (n=698) 100.3 0.68 (n=101)
101.2 0.3 (n=402)
Cortex (Cx) 100 0.26 (n=600) 100.5 0.2 (n=1029)
Fig. 5. Mean variations ( S.E.M.) of the in vivo 460 nm signal measured throughout the rat sleep-wake
cycle in the PAG-nRD area and in the cortex (1 count=10 s; n=number of counts). For the PAG-nRD, A
to E are single recording sites, in each of them measurements were performed during W, SWS and PS. It
must be noticed that in the PAG-nRD area the changes occurring in the NADH signal are very
heterogeneous. For the cortex (Cx), since the variations measured in each site were very homogeneous,
pooled values are reported. W=100 0.26 (n=600); SWS=100.5 0.2 (n=1029); PS=101.2 0.3 (n=402). It
can be underlined that in the Cx, contrary to the PAG-nRD, the changes in the NADH signal are
homogeneous. Statistics: an ANOVA followed by a multiple range test (Bonferroni–Dunnet) were used.
Comparisons shown in the figure (PAG-nRD and Cx) were performed according to SWS/W and PS/W;
significance of the differences is as follows: *P0.05; **P0.01; ***P0.001; (NS, non-significant
differences in E). Comparisons according to PS/SWS (not marked in the figure) are significant in B (*), C
(**) and D (***). Aq, aqueduct; see also other figures for abbreviations. Localizations of the sensor (with
reference to Paxinos and Watson atlas58) are in the frontal plane +1.00 for the PAG-nRD and +9.7 for the
Cx. The vigilance state of the animal, determined by polygraphic recordings, is noted at the bottom of
each histogram.

7. Brain NADH determination with an optic fibre sensor 689
during SWS or PS. It is also reported that cytosolic
PDH might be active in ACh synthesis75 which is
involved in PS executive mechanisms76 and also
released during this state of sleep.39 Such processes
take place during PS and are important for its
occurrence. Their contribution to the redox potential
is limited since for one AcCoA contributing to ACh
synthesis, 200 more are used in the tricarboxylic acid
cycle.75 It appears thus likely that the brain measure-
ments reported here may reflect changes occurring in
the redox state of the mitochondrial protein-bound
Fig. 6. Variations in the PAG-nRD 460 nm fluorescence NADH.
after a lethal dose of barbiturates (i.p.). Mean value of the
data was established during the 30 min period preceding the
injection (period referenced as 0%) and during the 15– Sleep data interpretation
30 min interval immediately following it (period referenced
as 100%). The curve fit equation is y=A (1-exp(-x/ ) and Measurements carried out inside the PAG-nRD
=4.31 min in this example. Each dot corresponds to the area under chronic conditions and reported here,
fluorescence pulses integrated for 6 s (digitizer full scale at
10 ns). (a), breathing cessation; see also other figures for indicate that variations of the 460 nm signal, occur-
abbreviations. ring throughout the sleep-wake cycle, are hetero-
geneous and dependent on the probe position and the
vigilance state. During SWS, according to the site
unperfused/perfused brain slices12 can rule out, in investigated, the signal height either increased signifi-
our physiological conditions, its significant partici- cantly or remained stable or exhibited a decreasing
pation in the signal generation through reflectance tendency. During PS, the changes observed were
and fluorescence. It is clear that haemoglobin more homogeneous and significant increases were
(oxygenated and deoxygenated) absorbs at 337 nm constantly observed. In the cortex, the signal showed
(excitation) and 460 nm (fluorescence). In this way, a constant tendency to increase during SWS and PS
the fluorescence intensity is influenced by tissue as compared to W. The fluorescence levels measured
blood contents. However, it can be emphasized that in this structure are, however, significant only during
the vascular bed is twice less dense in the nRD than PS/W. The variations in the redox potential appear,
in the cortex.18 It must be also noticed that these at first sight, difficult to interpret owing to the myriad
interferences are strongly amplified when the exper- of metabolic and transport processes regulating the
imental conditions do not allow self-homeostatic redox equivalent delivery to the mitochondria. Con-
regulation, as in the case with anaesthetized ani- siderable heterogeneity in the literature data3,19,71
mals. The present approach overcomes the exper- renders these mechanisms even more difficult to
imental artifacts attached to the animal preparation, interpret. In contrast with the in vitro preparations
since it was performed in the freely moving rat and in vivo anaesthetized animals, our results were
which self-regulates homeostatic aspects. obtained with animals in perfect physiological condi-
Concerning intra- and extracellular compart- tions in which all the parameters influencing the
ments, data obtained with cerebrospinal fluid (CSF) NAD+/NADH balance were self-regulated by the
indicate that this fluid does not yield a noticeable animal itself, i.e. oxygen supply, temperature regula-
460 nm fluorescence signal and is thus devoid of tion, food given ad libitum for the availability of basic
detectable amounts of extracellular NADH.55 It energetic substrates. Furthermore, brain NADH is
cannot be excluded, however, that CSF from brain closely linked with ATP production in the rat, mainly
cavities might differ from that contained in the from glucose and through the chain of electron
tissue. Anatomical and biochemical data, never- transfers (oxidative phosphorylation) in which
theless, lay stress on the fact that NADH is an oxygen is determinant.68 The balance of the redox
intracellular compound present mainly within potential depends on the oxidative phosphorylation
mitochondria.12,22,34,71 which favours the consumption of NADH, and the
Whether the autofluorescence is an index of the PDH and the tricarboxycylic acid cycles which both
free NADH and/or protein-bound NADH is another produce or regenerate NADH.3,13,23 This aspect is
issue. It is well-known that NADH fluorescence is clearly illustrated by the use of a lethal dose of
greatly enhanced by protein binding.27,41 A change in barbiturates which, in reducing the oxygen avail-
autofluorescence measurement can be interpreted as ability, limits the chemical processes at the AcCoA
a change in the redox state of NADH or a change in step and produces a considerable increase in the
its molecular environment (protein binding, mem- 460 nm NADH signal. During PS, generally the
brane effects...). With our subnanosecond time- NADH signal also increases in both areas investi-
resolved fluorescence sensor, we did not observe gated, i.e. the PAG-nRD and cortex, and the oxygen
significant differences in the subnanosecond time availability might again be the condition determinant
shapes of the NADH fluorescence derived either for such an effect. Several reports, indeed, add

8. 690 S. Mottin et al.
strength to such an hypothesis since hypoxia de- ties46,79 within the whole raphe nuclei. Throughout
creases PS.2,42 The increase observed in the signal the sleep-wake cycle, it is now well established that
intensity during this state of sleep thus reflects a the nRD 5-HT neurons exhibit a regular firing rate
higher activity of PDH and tricarboxcylic acid cycles during W, which decreases during SWS and even
directed towards an enhanced production of the more during PS.8,24,51 This pattern of discharge has
oxidative energy. Indeed, by this time, glucose and been referred to as PS-off.65 Furthermore, moderate
oxygen consumption increase,30 consumption of and localized cooling (10 C) of the nRD, which also
glycogen decreases36 while lactate concentrations re- produces a decrease in neuronal discharge, is sleep-
main unchanged.69 Thus, in our physiological condi- inducing.6,7 The iontophoretic application of 5-HT
tions and particularly during PS, an increase in the within the nRD decreases the discharge rate of 5-HT
NADH 460 nm signal would reflect an activation of neurons.73 During SWS and PS as compared to W,
the phosphorylative pathway in which metabolic these neurons are subjected to a somatodendritic
processes from glucose to ATP might be limited by release of 5-HT which, through an autoinhibitory
the oxygen availability. This view is again consist- process, decreases their discharge rate.10 The pres-
ently strengthened by the following three sets of data: ence of vesicles within 5-HT dendrites provides evi-
whether in the rat or in the cat there exists a rise in dence that 5-HT may be released from them.15,18
brain temperature during PS, accompanied by a fall This dendritic release might be triggered by non-
in cerebral blood flow at the beginning of the PS 5-HT axonal nerve endings impinging on the den-
episode,17,64 it is likely that this fall might limit the drites.10 Finally, in the structures where the 5-HT
oxygen availability; chloramphenicol, known to axonal nerve endings impinge (caudate, cortex, basal
strongly inhibit the first site of the oxidative phos- hypothalamus), the 5-HT release occurs throughout
phorylation,26 very efficiently reduces PS in the cat59 the sleep-wake cycle in an opposite manner, i.e.
and in the rat;14 glucose metabolism is clearly in- maximum during W and decreased during SWS and
creased during PS.44 Concerning the latter aspect it PS.10 Otherwise, the significant increase in glucose
can be emphasized that, whether in the cortex or in metabolism, reported to occur during PS,44 is per-
the PAG-nRD area, the magnitude of the index fectly in line with our results indicating that the
related to glucose utilization during PS as compared 460 nm NADH fluorescence is increased during PS.
to W44 is in good agreement with the NADH varia- It should be noticed that, paradoxically, when the
tions reported in the present paper. These variations serotoninergic neurons are silent, glucose metabolism
appear mild at first sight, but this is not surprising increases. The energy consumption might thus be
since they reflect the genuine variations occurring in a necessary for the processes attached to the dendritic
strict natural situation, i.e. alternation from W to PS. release of 5-HT which could occur through depolar-
Finally, according to the above discussion emphasiz- izing processes independent of the soma.43 In this
ing the importance of the oxidative energy for PS respect, it is well known that about 50% of the brain
occurrence, it is not excluded that its rate of produc- ATP is consumed by brain Na+, K+-ATPase and
tion and/or consumption might also influence its that dendrites require elevated Na+, K+-ATPase
periodicity. activity.23,33
The data obtained during SWS in specific PAG-
nRD sites or in the cortex, where the NADH signal
respectively decreases or exhibits non-significant
CONCLUSION
changes, indicate that the consumption of oxidative
energy is decreased, as currently suggested during this
state of sleep.30 However, the PAG-nRD probe loca- The adaptation of our FOCS in freely moving
tions in which the signal increases during SWS might, animals is now efficient for the evaluation of the laser
stimulated fluorescence. This procedure, providing
nevertheless, account for the existence of an oxidative
rapid assessment of the metabolic changes occurring
energy consumption during this state of sleep. In this
within defined brain regions with a good spatial
respect, a noticeable release of 5-HT has been re-
resolution, is of wide interest for biochemists, behav-
ported to take place within these sites during SWS.10
iourists and sleep physiologists. The evidence allow-
It should also be mentioned that the PAG-nRD area
ing one to suggest that the signal measured at 460 nm
contains other biochemical components which could
might be related to NADH has been discussed. This
require energy during SWS.21
signal varies according to the sleep states and gener-
ally increases during PS in the PAG-nRD area. The
Metabolism of the PAG-nRD area during paradoxical changes observed may reflect a state related to the
sleep oxidative energy.
The majority of the 5-HT perikarya is located
within the raphe nuclei (77.5%).79 The largest
Acknowledgements—This work was supported by INSERM
number is contained within the nRD (52% of the U52, DRET (Grant no 93-057), CNRS ERS-5645 and
raphe nuclei serotoninergic cells). These perikarya URA-842. We also thank Miss C. Limoges and Dr J.
also exhibit different morphological particulari- Carrew for improving the English text.

9. Brain NADH determination with an optic fibre sensor 691
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(Accepted 10 December 1996)