Effect of basal forebrain neuropeptide Y administration on sleep and spontaneous behavior in freely moving rats.
ABSTRACT Neuropeptide Y (NPY) is present both in local neurons as well as in fibers in the basal forebrain (BF), an area that plays an important role in the regulation of cortical activation. In our previous experiments in anaesthetized rats, significant EEG changes were found after NPY injections to BF. EEG delta power increased while power in theta, alpha, and beta range decreased. The aim of the present experiments was to determine whether NPY infusion to BF can modulate sleep and behavior in freely moving rats. In this study, microinjections were made into the BF. Saline was injected to the control side, while either saline or one of two doses of NPY (0.5 microl, 300-500 pmol) to the treated side. EEG as well as behavioral changes were recorded. Behavioral elements after the NPY injections changed in a characteristic fashion in time and three consecutive phases were defined. In phase I (half hour 2), activated behavioral items (moving, rearing, grooming) appeared frequently. In phase II (half hours 3 and 4) activity decreased, while motionless state increased. Reappearance of activity was seen in phase III (half hours 5 and 6). NPY injections caused sleep-wake changes. The three phases described for behavioral changes were also reflected in the sleep data. During phase I, lower NPY dose increased wakefulness and decreased deep sleep. Reduced behavioral activity seen in phase II was partially reflected in the sleep. In this phase, wakefulness tended to increase in the third half hour, while decreased in the 4th half hour. Deep sleep and total slow wave sleep non-significantly decreased in the third and increased in the 4th half hour. In most cases, wakefulness was elevated again during Phase III, while sleep decreased. Length of single sleep-wake epochs did not change after NPY injections. Our results suggest a role for NPY in the integration of sleep and behavioral stages via the BF.
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ABSTRACT: Spontaneous firing and behavior-related changes in discharge profiles of basal forebrain (BF) neurons are well documented, albeit the mechanisms underlying the variety of activity modes and intermodal transitions remain elusive. With the use of cell-attached recordings, this study identifies a range of spiking patterns in diagonal band Broca (DBB) noncholinergic cells of rats and tentatively categorizes them into low-rate random, tonic, and cluster firing activities. It demonstrates further that the multiplicity of discharge profiles is sustained intrinsically and persists after blockade of glutamate-, glycine/GABA-, and cholinergic synaptic inputs. Stimulation of muscarinic receptors, blockade of voltage-gated Ca(2+)-, and small conductance (SK) Ca(2+)-activated K(+) currents as well as chelating of intracellular Ca(2+) concentration accelerate low-rate random and tonic firing and favor transition of neurons into cluster firing mode. A similar trend towards higher discharge rates with switch of neurons into cluster firing has been revealed by activation of neuropeptide Y (NPY) receptors with the NPY or NPY(1) receptor agonist [Leu(31),Pro(34)]-NPY. Whole cell current-clamp analysis demonstrates that the variety of spiking modes and intermodal transitions could be induced within the same neuronal population by injection of bias depolarizing or hyperpolarizing currents. Taken together, these data demonstrate the intrinsic and highly variable character of regenerative firing in BF noncholinergic cells, subject to powerful modulation by classical neurotransmitters, NPY, and small membrane currents.Journal of Neurophysiology 04/2012; 108(2):406-18. · 3.30 Impact Factor
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ABSTRACT: Orexin A and orexin B are neuropeptides produced by a group of neurons located in the lateral hypothalamus which send widespread projections virtually to the whole neuraxis. Several studies indicated that orexins play a crucial role in the sleep-wake regulation and in the pathomechanism of the sleep disorder narcolepsy. As no data are available related to the EEG effects of orexin A in healthy, freely moving rats, the aim of the present experiments was to analyze EEG power changes in the generally used frequency bands after intracerebroventricular orexin A administration.Orexin A administration (0.84 and 2.8 nM/rat) differently affected fronto-occipital EEG waves in the different frequency bands recorded for 24 hours. Delta (1-4 Hz) and alpha (10-16 Hz) power decreased, while theta (4-10 Hz) and beta (16-48 Hz) power increased. Decrease of the delta power was followed by a rebound in case of the higher orexin A dose. This complex picture might be explained by the activation of several systems by the orexin A administration. Among these systems, cortical and thalamic circuits as well as the role of the neurons containing corticotrophin-releasing factor might be of significant importance.Acta Physiologica Hungarica 09/2012; 99(3):332-43. · 0.88 Impact Factor
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ABSTRACT: Sleep is important for maintenance of normal physiology in animals. In mammals, neuropeptide Y (NPY), a homolog of Drosophila neuropeptide F (NPF), is involved in sleep regulation, with different effects in human and rat. However, the function of NPF on sleep in Drosophila melanogaster has not yet been described. In this study, we investigated the effects of NPF and its receptor-neuropeptide F receptor (NPFR1) on Drosophila sleep. Male flies over-expressing NPF or NPFR1 exhibited increased sleep during the nighttime. Further analysis demonstrated that sleep episode duration during nighttime was greatly increased and sleep latency was significantly reduced, indicating that NPF and NPFR1 promote sleep quality, and their action on sleep is not because of an impact of the NPF signal system on development. Moreover, the homeostatic regulation of flies after sleep deprivation was disrupted by altered NPF signaling, since sleep deprivation decreased transcription of NPF in control flies, and there were less sleep loss during sleep deprivation and less sleep gain after sleep deprivation in flies overexpressing NPF and NPFR1 than in control flies, suggesting that NPF system auto-regulation plays an important role in sleep homeostasis. However, these effects did not occur in females, suggesting a sex-dependent regulatory function in sleep for NPF and NPFR1. NPF in D1 brain neurons showed male-specific expression, providing the cellular locus for male-specific regulation of sleep by NPF and NPFR1. This study brings a new understanding into sleep studies of a sexually dimorphic regulatory mode in female and male flies.PLoS ONE 01/2013; 8(9):e74237. · 3.73 Impact Factor
Brain Research Bulletin 72 (2007) 293–301
Effect of basal forebrain neuropeptide Y administration on sleep
and spontaneous behavior in freely moving rats
Attila T´ otha, T¨ unde Hajnika, L´ aszl´ o Z´ aborszkyb, L´ aszl´ o D´ et´ aria,∗
aDepartment of Physiology and Neurobiology, E¨ otv¨ os Lor´ and University, P´ azm´ any P´ eter S´ et´ any 1/C, H-1117 Budapest, Hungary
bCenter for Molecular and Behavioral Neuroscience, Rutgers University, Newark, New Jersey, USA
Received 6 July 2006; received in revised form 17 November 2006; accepted 9 January 2007
Available online 2 February 2007
Neuropeptide Y (NPY) is present both in local neurons as well as in fibers in the basal forebrain (BF), an area that plays an important role in
the regulation of cortical activation. In our previous experiments in anaesthetized rats, significant EEG changes were found after NPY injections
to BF. EEG delta power increased while power in theta, alpha, and beta range decreased. The aim of the present experiments was to determine
whether NPY infusion to BF can modulate sleep and behavior in freely moving rats.
In this study, microinjections were made into the BF. Saline was injected to the control side, while either saline or one of two doses of NPY
(0.5?l, 300–500pmol) to the treated side. EEG as well as behavioral changes were recorded.
Behavioral elements after the NPY injections changed in a characteristic fashion in time and three consecutive phases were defined. In phase
I (half hour 2), activated behavioral items (moving, rearing, grooming) appeared frequently. In phase II (half hours 3 and 4) activity decreased,
while motionless state increased. Reappearance of activity was seen in phase III (half hours 5 and 6).
NPY injections caused sleep–wake changes. The three phases described for behavioral changes were also reflected in the sleep data. During
phase I, lower NPY dose increased wakefulness and decreased deep sleep. Reduced behavioral activity seen in phase II was partially reflected in
the sleep. In this phase, wakefulness tended to increase in the third half hour, while decreased in the 4th half hour. Deep sleep and total slow wave
sleep non-significantly decreased in the third and increased in the 4th half hour. In most cases, wakefulness was elevated again during Phase III,
while sleep decreased. Length of single sleep–wake epochs did not change after NPY injections.
Our results suggest a role for NPY in the integration of sleep and behavioral stages via the BF.
© 2007 Elsevier Inc. All rights reserved.
Keywords: Neuropeptide Y; Cortical activation; Basal forebrain; Freely moving rats; Spontaneous behavior; Sleep; EEG
Neuropeptide Y (NPY) is one of the most abundant and
widely distributed neuropeptide in the mammalian central and
peripheral nervous system . It is involved in several physi-
ological functions such as feeding , memory , regulation
of blood pressure , circadian rhythms , and possibly in
the regulation of sleep–wake stages. NPY has anxiolytic-like
action in various animal anxiety models [10,14,40], but other
∗Corresponding author. Tel.: +361 381 2181; fax: +361 381 2182.
E-mail address: email@example.com (L. D´ et´ ari).
tion of catalepsy , increase in searching behavior , and
reduction in rearing activity  has been also reported after
intracerebroventricular (icv) administration.
NPY is present in the basal forebrain (BF), an area that plays
an important role in the regulation of cortical activation (for
a recent review, see ). The BF contains a heterogeneous
population of cholinergic and non-cholinergic (GABAergic,
peptidergic, and glutamatergic) corticopetal neurons as well
as various types of interneurons containing different peptides,
including NPY and somatostatin .
The role of NPY containing BF neurons in the modulation
of sleep–wake states and cortical EEG is not fully under-
stood. NPY was found to colocalize with GABA in many
forebrain neurons . Single NPY neurons can innervate
several cholinergic corticopetal neurons in the horizontal limb
of the diagonal band [37,43] and in the substantia innominata
0361-9230/$ – see front matter © 2007 Elsevier Inc. All rights reserved.
A. T´ oth et al. / Brain Research Bulletin 72 (2007) 293–301
 with symmetrical synapses  that are assumed to
be inhibitory . In anesthetized rats, NPY containing BF
neurons represent a subpopulation of S-cells (slow-wave-active
cells) which are silent during spontaneous or tail pinch-induced
cortical activation, but have a higher firing rate during episodes
of cortical slow waves . These neurons have been suggested
to inhibit cortically projecting cholinergic neurons . In our
previous experiments, significant changes were found in the
ipsilateral fronto-parietal EEG after NPY injections into the BF
of urethane-anaesthetized rats . EEG delta power increased,
while power in higher frequency ranges (theta, alpha, and beta)
decreased. These observations suggest that basal forebrain
NPY might have a role in the regulation of cortical activation.
The aim of the present experiments was to examine whether
NPY infusion into the BF of freely moving rats can modulate
sleep–wake stages and behavior.
2.1. Surgical procedure
Adult male Wistar rats (n=9, 265–295g) were used in the experiments. The
animals were anesthetized with sodium pentobarbital (Nembutal, 40mg/kg ip,
and lambda at the same horizontal plane . To record EEG activity, 0.8mm
the frontal (Br 2.0; L2.0) and parietal cortices (Br-4.5; L2.0) on both sides. An
California Fine Wire, CA, USA) were inserted into the neck musculature close
to the caudal surface of the skull. Electrode leads were soldered to a minia-
ture female connector. Stainless steel guide cannulas (C313G/SPC, 22 gauge,
Plastic One) with fitted dummy stylus (C313DC, 28 gauge, Plastics One) cut
1mm longer than the guide was lowered into BF on both sides in 10oangle to
enable intraparenchymal injections. The cannulas and the connector were fixed
to the bone with cranioplastic cement (Plastic One). Following surgery, animals
were returned to their home cages. Experiments were carried out in accordance
with the European Communities Council Directive (86/609/EEC) and with the
guidelines set forth in the US Public Health Service manual “Humane Care and
Use of Laboratory Animals” and the “Guide for the Care and Use of Labora-
tory Animals” (National Institutes of Health Guide). All efforts were made to
minimize animal suffering and the number of animals used.
2.2. Electrophysiological recording
Rats were housed in individual cages located a sound-attenuated room
cylinders (height: 330mm, diameter: 300mm). The whole setup was similar to
that described by Bertram et al. . Water and standard laboratory chow was
given ad libitum. The rats were exposed to a 12-h light/12-h dark cycle (lights
on at 09:30h).
Recording sessions started after an at least 2-week long recovery period
following surgery. All recordings were carried out in the home cages between
10:00a.m. and 8:30a.m. on the next day (22.5h). Rats were connected to the
recording system through flexible flat cables attached to fixed swivels (Plastic
One or Litton) above the home cages. The animals were left connected for the
whole experiment, except for brief periods of drug treatments.
EEG was measured between the frontal and parietal electrode pairs
on both sides (FPL-fronto-parietal left, FPR-fronto-parietal right) through
home-designed headstages based on the TLC2264I (Texas Instruments, USA)
amplifiers built into the male connector . EEG and EMG signals were
amplified, filtered (0.3–100Hz) and continuously digitalized by a pair of
computer and controlled by a custom-written software. Sampling rate was set to
102.4Hz to yield 512 data points per 5s recording time to facilitate Fast Fourier
Transformation (FFT). All EEG and EMG data obtained during the recording
sessions were stored on hard disk for off-line analysis.
2.3. Behavioral recording
Behavior was recorded in the first four hours of the sessions by commercial
video equipments. Videotapes were analyzed off-line by observers blind to the
treatment received by the animals. Using custom-written software, they scored
behavioral items into one of the following categories: quiet (absence of loco-
motion or movement of body parts), moving (locomotion or movement of body
parts), grooming, rearing, drinking, and eating. Time spent with the different
behavioral items was summarized for consecutive half hours.
Rats were placed into small, open boxes during injections and were gen-
tly restrained to prevent escaping if needed. Dummy cannulas were replaced
by 28 gauge internal cannulas (C313I/SPC, Plastics One) connected through
polyethylene tubing to microprocessor controlled syringe pumps (IITC Inc.,
CA, USA) holding two microsyringes (Hamilton, 25?l). Pressure injections
(volume 0.5?l, speed 0.25?l/min) were carried out bilaterally. Cannulas were
left in place for an additional 2min following injection. Rats were habituated
to the injection procedure before treatments started. We did not observe visible
signs of stress response during the injection procedures.
NPY (Sigma–Aldrich, Schnelldorf, Germany) was dissolved in sterile phys-
left side always received saline, while to the right side either saline (control) or
one of the two NPY doses was injected. Each rat received all three treatments
in a randomized order. Treatments were separated by at least 2 days.
2.5. Data analysis
To separate sleep stages, power spectra were constructed for consecutive 5-s
epochs from the EEG signals using a custom-made software. From the spec-
tra, integrated power of the following frequency bands was calculated: low
delta (0.5–2Hz), high delta (2–4Hz), total delta (0.5–4Hz), theta (4–10Hz),
alpha (10–16Hz), beta (16–30Hz), and gamma (30–48Hz). In addition, ratio
of theta and total delta bands were also determined. EMG power was integrated
between 5 and 48Hz. All these values were stored and used in the next steps
of the analysis. EEG recordings were 22.5h long, but only the first 4h were
Sleep stages were scored with the help of an interactive, semiautomatic
computer program using EEG data from the control (FPL, saline-injected) side.
(paradoxical sleep) epochs were also marked manually, by inspecting the calcu-
lated power values, the theta/delta ratio and the original EEG/EMG recordings.
Only PS epochs longer than 30s were included in the analysis.
EEG slow wave content (delta power, 0.5–4Hz) is closely and inversely
related to the level of cortical arousal , thus this parameter was used for
an approximate discrimination of active (AW) and quiet (QW) wakefulness,
light (LS) and deep (DS) sleep, as described earlier [6,8]. Briefly, delta power
histograms were constructed from the control recording taken after saline treat-
ment, excluding PS epochs. Five-second periods with delta power values falling
into the uppermost quarter (above 75% percentile) of the histogram were cate-
first quarter of the histogram were identified as LS, QW, and AW, respectively.
Delta power values limiting the four quarters of the histogram were then used
as criteria for scoring all the other recordings. Total wakefulness (tWAKE; AW
and QW together) as well as total slow wave sleep (tSWS; LS and DS together)
were also calculated.
2.6. Statistical analysis
The first half hour was left out, as rats needed time to calm down follow-
ing the injection procedure. Time spent with a given behavioral element was
A. T´ oth et al. / Brain Research Bulletin 72 (2007) 293–301
summarized for consecutive half hours and expressed as percent of the period.
Sleep–wake stages were similarly summarized, but in this case the number of
epochs for the different sleep–wake states was also calculated. Statistical sig-
nificance of the observed changes was checked with two-way mixed-design
ANOVA (split-plot) with time as the first, and NPY dose as the second fac-
tor. All tests were two-tailed and p<0.05 was accepted as the lowest limit of
urethane (1.2g/kg, ip). To assess the spread of injected peptides in the BF and
to verify the cannula locations, horseradish peroxidase (HRP; Sigma–Aldrich,
by 400ml of 4% paraformaldehyde in PBS (pH 7.4) immediately after the HRP
injection to prevent uptake and cellular transport. Brains were removed and
cryoprotected in the same fixative containing 30% sucrose until equilibration.
Coronal sections (50?m) were cut through the area of interest with a freezing
The peroxidase reaction was visualized with 3,3?-diaminobenzidine (DAB;
Sigma–Aldrich, Schnelldorf, Germany) as the chromogen. The spread of HRP
was found to be about 1000?m from the injection site. Sections were coun-
terstained with gallocyanine (Sigma, Germany), dehydrated, and coverslipped
with DepEx (Serva, Heidelberg, Germany). Injection sites were located based
on the stereotaxic atlas of Paxinos and Watson .
3.1. Behavioral changes
Rats tolerated handling and administration of drugs with-
out intensive struggling or vocalization. However, the injection
procedure caused a transient, non-specific behavioral activa-
tion gradually wearing down during the first half hour after the
injection. This period was excluded from behavioral as well
as from sleep analysis. Following this period, the motionless
state dominated the behavior of rats, representing in average
at least 60% of total recording time. However, moderate dif-
ferences in the proportion of the quiet state in the control as
well as in all other recording sessions enabled the definition
of three consecutive phases (Fig. 1). Phase I (half hour 2) was
characterized by a relatively high amount of activated behav-
ioral items (moving, rearing, grooming) probably still due to
the aftereffects of injection. In phase II (half hours 3 and 4)
activity sharply decreased, sleep increased. This period was fol-
lowed by the reappearance of activity in phase III (half hours 5
and 6). In general, NPY caused only mild to moderate changes
in behavior that often remained below the p<0.05 significance
lower dose significantly increased grooming too. However, the
60% and 15% of the total time, respectively (Fig. 1A and B).
Phase II. Moving, rearing, grooming, and eating strongly
decreased in half hour 3 and remained low in half hour 4 as
well. At the same time, quiet, motionless state increased after
saline as well as NPY treatments. As rats spent most of the time
motionless and other behavioral elements were only sporadic,
very few significant changes were seen. NPY treatment tended
to decrease movements and increase quiet state. This effect was
more pronounced with the lower dose and reached significant
levels in half hour 4. Drinking occurred less frequently after any
Fig. 1. Behavioral effects of NPY injected to the BF (n=8). (A) Moving, (B) quiet, (C) rearing, (D) grooming, (E) eating and (F) drinking. The proportion of the
quiet state changed in a characteristic fashion in the control as well as in all other recordings enabling the definition of three consecutive phases (phase I: half hour 2,
phase II: half hours 3 and 4, phase III: half hours 5 and 6). NPY and saline injections were made at the beginning of the light period and behavior was videotaped for
4h. The first half hours after the injections were excluded from the analysis. After the excluded period, values scored as a given behavioral element were summarized
from the consecutive 30min long periods and expressed as percent of the period. Asterisk (*) indicates significant deviation from the corresponding control (saline)
value after the NPY injections. Significance was tested with two-way mixed-design ANOVA (split-plot). Significance levels:*p<0.05;**p<0.01;***p<0.001. Data
are expressed as mean and S.E.M.
A. T´ oth et al. / Brain Research Bulletin 72 (2007) 293–301
of the NPY doses compared to saline injection (Fig. 1F). This
difference might have reflected some rebound effect, as during
Phase I, treatment groups drank more frequently than controls.
Phase III. Following the behavioral inactivity in half hours
3 and 4, rats became more active again, regardless of the treat-
ment. The lower NPY dose significantly decreased time spent
motionless and increased grooming, eating and drinking in half
hour 5. Grooming was still high in half hour 6 (Fig. 1D). The
higher dose caused less significant changes in behavior, though
it strongly increased grooming and eating in half hour 6.
3.2. Sleep changes
NPY injections into the basal forebrain caused no abnor-
mal EEG activity and only moderate changes in the sleep–wake
states (Figs. 2 and 3). The three consecutive phases described
proportion of sleep–wake stages as well.
Phase I. Half hour 2 following treatment was characterized
by behavioral activation. The lower dose of NPY increased
the amount of wakefulness (Fig. 2C) and decreased deep sleep
(Fig. 3D). Similar, but much weaker effects were seen after the
higher dose. In this case, changes were not statistically signifi-
cant. The amount of light sleep and paradoxical sleep remained
unchanged (Fig. 3A and B).
During phase II, behavioral activation was strongly reduced.
However, this decrease of activity was not so evident in the
non-significant elevation in the third half hour in case of both
doses, while decrease was seen in the 4th half hour (Fig. 2). DS
in the 4th half hour (Fig. 3C and D). Higher dose significantly
increased PS in the 4th half hour (Fig. 3A).
phase III, while sleep values (LS, DS, PS, tSWS) decreased
(Fig. 3). However, the higher NPY dose significantly increased
DS and tSWS in the 5th half hour (Fig. 3C and D). After phase
III, sleep decreased and wakefulness increased until the end of
the light phase regardless of the treatment.
The observed changes in the total amount of time spent in
different sleep stages resulted from the alteration of the num-
ber of episodes (data not shown). Thus, NPY injections had no
influence on the length of single sleep–wake epochs.
3.3. Histological results
As Fig. 4 depicts injection sites were located in the ventral
part of globus pallidus, substantia innominata, ventral pal-
lidum and magnocellular preoptic nucleus, BF regions that
were found to contain large numbers of cholinergic cells by
Fig. 2. Effects of NPY injected to the BF on wakefulness (n=6). The figure shows % deviation from the control and S.E.M. (A) Active wakefulness, (B) quiet
wakefulness, (C) total wakefulness (active and quiet wakefulness together), (D) absolute total wakefulness values after control (saline) injections. NPY and saline
injections were made at the beginning of the light period. The first half hour after the injections were excluded from the analysis. After the excluded period, data
were analyzed in 30min long blocks. Asterisk (*) indicates significant deviation from the corresponding control (saline) value after the NPY injections. Significance
was tested with two-way mixed-design ANOVA (split-plot). Significance levels:*p<0.05;**p<0.01;***p<0.001.
A. T´ oth et al. / Brain Research Bulletin 72 (2007) 293–301
Fig. 3. Effects of NPY injected to the BF on sleep (n=6). The figure shows % deviation from the control and S.E.M. (A) Paradoxical sleep, (B) light sleep, (C) deep
sleep, (D) total slow wave sleep (light and deep sleep together), (E) absolute total slow wave sleep values after control (saline) injections. NPY and saline injections
were made at the beginning of the light period. The first half hour after the injections were excluded from the analysis. After the excluded period, data were analyzed
in 30min long blocks. Blank asterisk (?) indicates significant deviation from the corresponding control (saline) value after the injection of the lower NPY dose
(300pmol/0.5?l). Asterisk (*) indicates significant deviation from the corresponding control (saline) value after the NPY injections. Significance was tested with
two-way mixed-design ANOVA (split-plot). Significance levels:*p<0.05;**p<0.01;***p<0.001.
previous anatomical studies [24,25]. There were no systematic
differences in NPY effects depending on the exact site of the
injection (data not shown). Fig. 5 shows a photomicrograph of
a representative cannula location in the BF.
ioral effects of NPY injected to the BF in freely moving rats. In
our previous experiments in urethane-anaesthetized rats, NPY
injection to BF increased delta power and decreased power in
higher frequency ranges (theta, alpha and beta) in the ipsilat-
eral EEG . We attributed these changes to a local inhibitory
effect of NPY on corticopetal neurons, and expected a decrease
tions in freely moving rats. In contrast, changes were more
complex, but a tendency toward more wakefulness and more
state were observed, especially following the lower dose.
NPY was found to influence spontaneous behavior in several
studies after icv or intraparenchymal administration into dif-
ferent brain regions. NPY effects clearly depended on the site