The structure of the dorsal raphe nucleus and its relevance to the regulation of sleep and wakefulness

Article (PDF Available)inSleep Medicine Reviews 14(5):307-17 · February 2010with24 Reads
DOI: 10.1016/j.smrv.2009.11.004 · Source: PubMed
Abstract
Serotonergic (5-HT) cells in the rat dorsal raphe nucleus (DRN) appear in topographically organized groups. Based on cellular morphology, expression of other neurotransmitters, afferent and efferent connections and functional properties, 5-HT neurons of the DRN have been grouped into six cell clusters. The subdivisions comprise the rostral, ventral, dorsal, lateral, caudal and interfascicular parts of the DRN. In addition to 5-HT cells, neurons containing γ-aminobutyric acid (GABA), glutamate, dopamine, nitric oxide and the neuropeptides corticotropin-releasing factor, substance P, galanin, cholecystokinin, neurotensin, somatostatin, vasoactive intestinal peptide, neuropeptide Y, thyrotropin-releasing hormone, growth hormone, leu-enkephalin, met-enkephalin and gastrin have been characterized in the DRN. Moreover, numerous brain areas have neurons that project to the DRN and express monoamines (norepinephrine, histamine), amino acids (GABA, glutamate), acetylcholine or neuropeptides (orexin, melanin-concentrating hormone, corticotropin-releasing factor and substance P) that directly or indirectly, through local circuits, regulate the activity of 5-HT cells. The 5-HT cells predominate along the midline of the rostral, dorsal and ventral subdivisions of the DRN and outnumber the non-5-HT cells occurring in the raphe nucleus. The GABAergic and glutamatergic neurons are clustered mainly in the lateral and dorsal subdivisions of the DRN, respectively. The 5-HT(1A) receptor is located on the soma and the dendrites of 5-HT neurons and at postsynaptic sites (outside the DRN). It is expressed, in addition, by non-5-HT cells of the DRN. The 5-HT(1B) receptor is located at presynaptic and postsynaptic sites (outside the boundaries of the DRN). It has been described also in the ventromedial DRN where it is expressed by non-5-HT cells. The 5-HT(2A) and 5-HT(2C) receptors are located within postsynaptic structures. At the level of the DRN the 5-HT(2A) and 5-HT(2C) receptor-containing cells are predominantly GABAergic interneurons and projection neurons. Within the boundaries of the DRN the 5-HT(3) receptor is expressed by, among others, glutamatergic interneurons. 5-HT(7) receptors in the DRN are not localized to serotonergic neurons but, at least in part, to GABAergic cells and terminals. The complex structure of the DRN may have important implications for neural mechanisms underlying 5-HT modulation of wakefulness and REM sleep.
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PHYSIOLOGICAL REVIEW
The structure of the dorsal raphe nucleus and its relevance to the regulation
of sleep and wakefulness
Jaime M. Monti
*
Department of Pharmacology and Therapeutics, School of Medicine Clinics Hospital, Montevideo 11600, Uruguay
article info
Article history:
Received 16 September 2009
Received in revised form
15 November 2009
Accepted 16 November 2009
Available online 12 February 2010
Keywords:
Serotonin
Brain stem
Sleep
Waking
REM sleep
Dorsal raphe nucleus
Laterodorsal and pedunculopontine
tegmental nuclei
5-HT receptors
summary
Serotonergic (5-HT) cells in the rat dorsal raphe nucleus (DRN) appear in topographically organized
groups. Based on cellular morphology, expression of other neurotransmitters, afferent and efferent
connections and functional properties, 5-HT neurons of the DRN have been grouped into six cell
clusters. The subdivisions comprise the rostral, ventral, dorsal, lateral, caudal and interfascicular parts of
the DRN. In addit ion to 5-HT cells, neurons containing
g
-aminobutyric acid (GABA), glutamate, dopa-
mine, nitric oxide and the neuropeptides corticotropin-releasing factor, substance P, galanin, chole-
cystokinin, neurotensin, somatostatin, vasoactive intestinal peptide, neuropeptide Y, thyrotropin-
releasing hormone, growth hormone, leu-enkephalin, met-enkephalin and gastrin have been charac-
terized in the DRN. Moreover, numerous brain areas have neurons that project to the DRN and express
monoamines (norepinephrine, histamine), amino acids (GABA, glutamate), acetylcholine or neuropep-
tides (orexin, melanin-concentrating hormone, corticotropin-releasing factor and substance P) that
directly or indirectly, through local circuits, regulate the activity of 5-HT cells. The 5-HT cells predom-
inate along the midline of the rostral, dorsal and ventral subdivisions of the DRN and outnumber the
non-5-HT cells occurring in the raphe nucleus. The GABAergic and glutamatergic neurons are clustered
mainly in the lateral and dorsal subdivisions of the DRN, respectively. The 5-HT
1A
receptor is located on
the soma and the dendrites of 5-HT neurons and at postsynaptic sites (outside the DRN). It is expressed,
in addition, by non-5-HT cells of the DRN. The 5-HT
1B
receptor is located at presynaptic and post-
synaptic sites (outside the boundaries of the DRN). It has been described also in the ventromedial DRN
where it is expressed by non-5-HT cells. The 5-HT
2A
and 5-HT
2C
receptors are located within post-
synaptic structures. At the level of the DRN the 5-HT
2A
and 5-HT
2C
receptor-containing cells are
predominantly GABAergic interneurons and projection neurons. Within the boundaries of the DRN the
5-HT
3
receptor is expressed by, among others, glutamatergic interneurons. 5-HT
7
receptors in the DRN
are not localized to serotonergic neurons but, at least in part, to GABAergic cells and terminals. The
complex structure of the DRN may have important implications for neural mechanisms underlying 5-HT
modulation of wakefulness and REM sleep.
Ó 2009 Elsevier Ltd. All rights reserved.
Introduction
Serotonin (5-HT) was initially proposed to be responsible for the
initiation and maintenance of slow wave sleep (SWS).
1
However,
a series of findings from several laboratories seriously challenged
the serotonergic hypothesis of sleep. Thus, the firing rate of sero-
tonin-containing dorsal raphe nucleus (DRN) neurons decreases
during SWS relative to waking (W)
2
; 5-HT release in many brain
regions occurs predominantly during W, and diminishes at its
lowest level during rapid-eye-movement sleep (REMS)
3
5,7-dihydroxytryptamine, which produces a selective and exten-
sive depletion of brain 5-HT, does not significantly affect SWS
4
; and
there is no significant difference between tryptophan-deficient and
replete rats in time spent in SWS.
5
In regard to the alternative
hypothesis,
6
there is no firm evidence to support the proposal that
5-HT released during W might act as a neurohormone and induce
the synthesis and/or release of hypnogenic factors secondarily
responsible for SWS and REMS occurrence. Based on neurochem-
ical, electrophysiological, genetic and neuropharmacological
approaches, it is presently accepted that 5-HT functions to promote
W and to inhibit REMS.
This two-part review attempts: 1. to describe the neurotrans-
mitters found in each of the subdivisions of the DRN as well as the
corresponding afferent and efferent connections; 2. to discuss the
*
Tel.: þ598 2 710 58 07.
E-mail address: jmonti@mednet.org.uy
Contents lists available at ScienceDirect
Sleep Medicine Reviews
journal homepage: www.elsevier.com/locate/smrv
1087-0792/$ see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.smrv.2009.11.004
Sleep Medicine Reviews 14 (2010) 307–317
Author's personal copy
role of DRN serotonergic and non-serotonergic neurons, and of
their receptors, in regulating the behavioral state, and 3. to raise
questions that will encourage further research.
Neural structures and neurotransmitters involved in the
regulation of sleep and waking
The neural structures involved in the promotion of W are
located in the brain stem, the hypothalamus, and the basal fore-
brain (BFB). The nuclei found in the brain stem include, among
others, serotonin [5-HT: DRN, median raphe nucleus (MRN)]-con-
taining neurons.
Neurons in the preoptic area, anterior and lateral hypothalamus,
and adjacent BFB constitute the sleep-inducing system.
7
A majority
of these neurons contain
g
-aminobutyric acid (GABA), galanin and
melanin-concentrating hormone (MCH), three inhibitory neuro-
transmitters, and project to the DRN.
Cholinergic neurons of the laterodorsal and pedunculopontine
tegmental nuclei (LDT/PPT) act to promote REMS. All these neurons
are inhibited by serotonergic, noradrenergic (NE), histaminergic
(HA), orexinergic (OX) and dopaminergic (DA) cells.
8,9
The release of
5-HT and other neurotransmitters in areas relevant to REMS occur-
rence has been shown to be abundant during W, whereas during
REMS only acetylcholine (ACh) is released at a significant rate.
10
The structure of the DRN
The serotonin-containing neurons of the DRN provide the
principal source of serotonergic innervation of the telencephalon,
diencephalon, mesencephalon, and rhombencephalon of laboratory
animals and man.
11,12
In this respect, most of the serotonergic
innervation of the cerebral cortex, amygdala, BFB, thalamus, pre-
optic and hypothalamic areas, raphe nuclei, locus coeruleus (LC),
and pontine reticular formation comes from the DRN. In turn, inputs
to the DRN 5-HT cells have been found from the cerebral cortex, the
limbic system, the BFB, the hypothalamus, and the cholinergic,
dopaminergic, and noradrenergic nuclei of the brain stem.
The DRN contains 5-HT and non-5-HT neurons. The latter
express a variety of substances including DA, GABA and glutamate
(GLU). In addition, nitric oxide (NO) and the neuropeptides corti-
cotropin releasing factor (CRF), substance P (SP), galanin,
cholecystokinin (CCK), neurotensin (NT), somatostatin (SOM),
vasoactive intestinal peptide (VIP), neuropeptide Y (NPY), thyro-
tropin-releasing hormone (TRH), growth hormone (GH) and
growth hormone-releasing hormone (GHRH), leu-enkephalin, met-
enkephalin, and gastrin have been characterized in the DRN. These
various neuropeptides are synthesized either by distinct cells in the
DRN or are coexpressed with other neurochemicals such as 5-HT
and DA. Moreover, numerous brain areas have neurons that project
to the DRN and express monoamines (NE, HA), amino acids (GABA,
GLU), ACh or neuropeptides (OX, MCH, CRF and SP among others)
that directly or indirectly, through local circuits, regulate the
activity of 5-HT cells.
13–22
Based on cellular morphology,
23
expression of other neuro-
transmitters,
16
afferent and efferent connections,
24,25
and functional
properties,
21
5-HT neurons of the DRN have been grouped into
several subnuclei or cell clusters. These differences among
subpopulations of 5-HT neurons may have important implications
for neural mechanisms underlying 5-HT modulation of sleep and
wakefulness. In addition, non-5-HTcells contribute to the regulation
of the activity of 5-HT neurons during the sleep-wake cycle through
local circuits and/or their mediation of the effects of afferent inputs.
Subdivisions of the DRN of the rat
Lowry et al.
22
have proposed six subdivisions of the DRN of the
rat. The subdivisions comprise: 1. the rostral part of the DRN
(DRNR) that contains medium-sized 5-HT cells, mainly in its ventral
portion. Neurons have been described also that contain DA, GABA,
GLU, NO, and the neuropeptides SP, CRF, NPY, leu-enkephalin, met-
enkephalin, CCK, NT, SOM and VIP.
26,27
The DRNR receives axons
from the hypothalamus,
28
and projects to the mesencephalon, basal
ganglia, and cerebral cortex
13,29,30
(Fig. 1); 2. the ventral part of the
DRN (DRNV) includes predominantly small, round 5-HT cells. In
addition, cells are present in the DRNV that contain GABA, galanin,
and NO.
22,31
The DRNV receives projections from the limbic system
and the cerebral cortex.
25,32
In turn, neurons of the DRNV project to
the basal ganglia and the cerebral cortex
30,33,34
(Fig. 2); 3. the dorsal
part of the DRN (DRND) comprises medium-sized or bipolar 5-HT
neurons. It includes, in addition, GABA, galanin, CRF, SOM, and NO-
containing cells.
20
The DRND receives fibers from the BFB and
cerebral cortex
25
and projects to the hypothalamus, limbic system,
Nomenclature
5-HT seratonin
ACh acetylcholine
BFB basal forebrain
CCK cholecystokinin
CRF corticotropin releasing factor
DA dopamine
DRN dorsal raphe nucleus
DRNC dorsal raphe nucleus caudal
DRND dorsal raphe nucleus dorsal
DRNI dorsal raphe nucleus interfascicular
DRNR dorsal raphe nucleus rostral
DRNV dorsal raphe nucleus ventral
DRNVL dorsal raphe nucleus ventrolateral
GABA
g
-aminobutyric acid
GH growth hormone
GHRH growth hormone-releasing hormone
GLU glutamate
HA histamine
LC locus coeruleus
LDT/PPT laterodorsal and pedunculopontine tegmental nuclei
MCH melanin-concentrating hormone
mPRF medial pontine reticular formation
MRN median raphe nucleus
NE norepinephrine
NO nitric oxide
NOS nitric oxide synthase
NPY neuropeptide Y
NT neurotensin
OX orexinergic
REMS rapid-eye-movement sleep
SNc substantia nigra pars compacta
SOM somatostatin
SP substance P
SWS slow wave sleep
TRH thyrotropin-releasing hormone
VIP vasoactive intestinal peptide
VLPAG ventrolateral periaqueductal gray
VTA ventral tegmental area
W wakefulness
J.M. Monti / Sleep Medicine Reviews 14 (2010) 307–317308
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BFB, and cerebral cortex
20,22,35
(Fig. 3); 4. the lateral wings of the
DRN include the ventrolateral part of the DRN (DRNVL) and the
adjacent ventrolateral periaqueductal gray (VLPAG). The DRNVL/
VLPAG comprises very large multipolar 5-HT neurons as well as
a great number of GABAergic cells.
36
The DRNVL/VLPAG receives
afferents from the mesencephalon, hypothalamus, limbic system
and BFB
37–39
and projects to the mesencephalon, hypothalamus,
and thalamus
40,41
(Fig. 4); 5. the caudal DRN (DRNC) contains
predominantly medium-sized 5-HT cells. In addition CCK,
enkephalin, NPY, SOM, and SP-containing cells have been charac-
terized in this cell cluster. The DRNC receives fibers from the
rhombencephalon, mesencephalon, hypothalamus, thalamus, and
cerebral cortex
42
and sends projections to the thalamus and limbic
system
12
(Fig. 5); 6. the interfascicular part of the DRN (DRNI)
includes spindle-shaped 5-HT cells.
43
Although afferents to the
subregion have not been characterized to date, projections from the
DRNI have been described that reach the thalamus, limbic system,
and cerebral cortex
44,45
(Fig. 6).
Ascending tracts that arise from DRN neurons
At least six different ascending tracts have been recognized by
Azmitia
43
(see Lowry et al.,
22
for review and references). The
ascending tracts include: 1. the dorsal raphe forebrain tract that
Figure 1. Afferent and efferent connections and neurotransmitters characterized in the rostral part of the dorsal raphe nucleus (DRNR). Sections according to Paxinos and
Watson
143
.
Figure 2. Afferent and efferent connections and neurotransmitters characterized in the ventral part of the dorsal raphe nucleus (DRNV).
J.M. Monti / Sleep Medicine Reviews 14 (2010) 307–317 309
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originates mainly from neurons that form the DRND region and
innervates the basal ganglia, amygdala and medial prefrontal
cortex (mPFC); 2. the median raphe forebrain tract that arises
mainly from the DRNI and the MRN and innervates the medial
septal nuclei, hippocampus, mPFC, and cingulate cortex; 3. the
dorsal raphe nucleus cortical tract that arises from the DRNR,
DRNV and DRND subregions and innervates the caudate-putamen
and the parieto-temporal cortex; 4. the dorsal raphe nucleus
periventricular tract that arises from 5-HT cells of the DRNC and
innervates the periventricular thalamic and hypothalamic regions;
5. the dorsal raphe arcuate tract that originates in the DRNVL/
VLPAG and innervates the substantia nigra compacta (SNc), the
ventrolateral geniculate body, and the suprachiasmatic nuclei; 6.
the raphe medial tract that originates from both the DRNR and
MRN and innervates the interpeduncular nucleus and the
mammillary body.
Of note, fibers that arise from neurons corresponding to the
arousal systems and project to the DRN maintain also a topographic
order with respect to afferent inputs. Accordingly, the orexinergic
system reaches both the dorsal (DRNV, DRND, DRNI) and the
median raphe nuclei; 2. the histaminergic and noradrenergic
system project primarily to the DRN. The latter reaches the DRND,
DRNVL, DRNC and DRNI; 3. the cholinergic system projects
predominantly to the MRN.
Figure 3. Afferent and efferent connections and neurotransmitters characterized in the dorsal part of the dorsal raphe nucleus (DRND).
Figure 4. Afferent and efferent connections and neurotransmitters characterized in the lateral wings of the dorsal raphe nucleus (ventrolateral part of the dorsal raphe nucleus and
adjacent ventrolateral periaqueductal gray) (DRNVL/VLPAG).
J.M. Monti / Sleep Medicine Reviews 14 (2010) 307–317310
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Neurons and fibers found in the DRN of adult rats
Serotonergic neurons and fibers
It has been estimated that two thirds of neurons found in the
DRN of adult rats are serotonergic.
16,46
The serotonergic cells are
present throughout the rostral-caudal extent of the DRN, in all
clusters of the nucleus. However, they predominate along the
midline of the rostral, dorsal and ventral subdivisions of the
DRN.
36,41,47
Recently, Yasufuku-Takano et al.
48
found that 62% of
cultured neurons of the DRN from postnatal 9–12-day-old rats
were serotonergic, and that ninety-two percent of the serotonergic
neurons responded to the 5-HT
1A
receptor agonist 8-OH-DPAT with
an increase in an inwardly rectifying K
þ
conductance. The findings
by Yasufuku-Takano et al.
48
support the proposal that 5-HT neurons
outnumber the non-5-HT cells occurring in the DRN.
GABAergic neurons and fibers
GABAergic neurons are abundant throughout the DRN of the rat.
Although most of the DRN subnuclei contain GABAergic interneu-
rons, they predominate in the lateral wings (DRNVL) of the raphe
Figure 5. Afferent and efferent connections and neurotransmitters characterized in the caudal dorsal raphe nucleus (DRNC).
Figure 6. Efferent connections and neurotransmitters characterized in the interfascicular part of the dorsal raphe nucleus (DRNI).
J.M. Monti / Sleep Medicine Reviews 14 (2010) 307–317 31 1
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nucleus.
18,36,49,50
In addition, GABAergic neurons located in several
neuroanatomical structures including the ventral pallidum, the
hypothalamus, the mesencephalon and the rhombencephalon,
project to the DRN.
51
A number of classes of GABA receptors such as
the GABA
A
, GABA
B
, and GABA
C
receptors have been characterized in
the central nervous system of several species. The GABA
A
receptor,
an ionotropic chloride channel, has a predominant role in the
modulation of sleep variables. It is made up of five heteromeric
subunits arranged around the ion channel. GABA
A
receptors have
been analyzed by gene knockout strategies and knock-in point
mutations. The sedative-hypnotic activity of most hypnotic drugs
has been shown to be dependent on the integrity of the
a
1
subunit.
52
The GABA
A
receptor is expressed by GABAergic inter-
neurons located in the DRN and GABAergic fibers projecting to the
nucleus. The GABA
B
receptor is a metabotropic G-protein coupled
receptor. Using microdialysis in vivo it has been determined that the
GABA
B
agonist R(þ)-baclofen enhances 5-HT output in the DRN of
the rat. The effect has been proposed to depend on the activation of
GABA
B
receptors located on GABAergic interneurons and inhibitory
afferents projecting to serotonergic neurons.
53
The relevance of the
GABA
C
receptor in sleep mechanisms is presently unknown.
Dopaminergic neurons and fibers
Dopaminergic neurons in the DRN are restricted to the rostral
half of the nucleus (DRNR)
36,54,55
and project to the mPFC, striatum,
nucleus accumbens, and lateral septal nuclei.
56
Glutamatergic neurons and fibers
A relatively small number of glutamatergic neurons have been
characterized in the DRNR.
57
Moreover, glutamatergic inputs to the
DRN have been described that originate in the mPFC, amygdala,
various hypothalamic areas, the parabrachial nuclei, and the LDT
among others.
42
Thus, it can be proposed that excitatory gluta-
matergic inputs from both cortical and subcortical areas, and glu-
tamatergic interneurons located in the DRN might be involved in
the serotonergic regulation of sleep and W.
Nitric oxide synthase (NOS)-containing neurons and fibers
Nicotinamide adenine dinucleotide phosphate diaphorase
(NADPH-diaphorase) histochemistry is a reliable marker for NOS,
the enzyme that catalyzes the synthesis of NO in the central
nervous system.
58
Johnson and Ma
17
(1993) and Wotherspoon
et al.
59
have established that numerous neurons in the DRN of the
rat contain both 5-HT-like immunoreactivity and NADPH-diapho-
rase activity. The majority of NOS positive neurons are clustered in
the dorsomedial and ventromedial (DRND and DRNV) cell groups of
the DRN, with only a limited number of NOS-positive cells in the
lateral groups (DRNVL/VLPAG). It should be mentioned that
according to Le
´
ger et al.
60
numerous NOS-synthesizing neurons
located in neuroanatomical structures involved in the regulation of
the behavioral state, including the preoptic area, lateral and
posterior hypothalamus, ventral tegmental area (VTA), and LDT/
PPT, send projections to the DRN of the rat.
CRF-containing neurons and fibers
CRF type 1 and type 2 receptors have been detected in the DRN.
The CRF1 receptor is expressed at low levels exclusively by
GABAergic cells whereas the CRF2 receptor is expressed at rela-
tively high levels by 5-HT and GABAergic neurons in the middle and
caudal subdivisions of the DRN, respectively.
36
Moreover, rodent studies have shown that CRF cell bodies
project from the paraventricular nucleus of the hypothalamus, the
central nucleus of the amygdala, and the bed nucleus of the stria
terminalis to the DRNI and the ventromedial subregion of the
DRN.
20,61
In this respect, CRF-immunoreactive fibers have been
detected in close association with 5-HT cells of the DRNI and the
DRNV.
62
CRF fibers have been found also to contact non-seroto-
nergic, mainly GABAergic, neurons in rat DRN.
63
In addition, CRF is
colocalized with 5-HT neurons predominantly in the DRNR and the
DRND.
20,63
SP-containing neurons and fibers
Substance P is widely distributed in the central nervous system
of mammals and exerts its functions by binding mainly to the
neurokinin-1 (NK
1
) receptor.
64
NK
1
receptors have been detected in
several brain structures related to the regulation of sleep and W
including the DRN.
65,66
In this respect, 5-HT cells endowed with
NK
1
receptors tend to predominate in the dorsomedial and the
caudal part of the DRN.
20,66
Of note, at the DRN level SP is coex-
pressed with 5-HT in approximately 50% of ascending serotonergic
neurons in the squirrel monkey. In contrast, this value is much
smaller in the cat and the rat.
18
Interestingly, the lateral habenula
nuclei of the diencephalon innervate the DRN via an inhibitory
projection, and experimental data from the rat indicate that their
lesion leads to decreased SP levels in the DRN.
67,68
On the other
hand, SP-containing neurons in the DRN of the rat have been found
to send projection fibers to the central amygdaloid nucleus, zona
incerta, and medial and lateral thalamic nuclei.
69
Galanin-containing neurons and fibers
Galanin, an inhibitory neuropeptide, is widely distributed in the
central nervous system, including the DRN. Galanin has been found
to be coexpressed with 5-HT in approximately 40% of the DRN cells
corresponding to the DRND and DRNV subdivisions.
22,70
The
neuropeptide is present, in addition, in afferents from the LC, pre-
optic/anterior hypothalamus, thalamus and BFB.
71
Galanin regu-
lates a number of behaviors through the activation of three
G-protein-coupled receptors, GalR1, GalR2 and GalR3.
72
However,
at the level of the DRN galanin binding sites correspond predomi-
nantly to GalR1 and GalR2 receptors that are expressed by 5-HT
cells and GABAergic interneurons.
73
VIP-containing neurons and fibers
Ahnaou et al.
74
have described VIP cells and nerve fibers
immunostained for VIP in the DRN of the rat. Moreover, neurons
immunoreactive to VIP in the DRNR have been shown to project to
the hypothalamus, thalamus, and bed nucleus of the stria termi-
nalis.
75–77
CCK-containing neurons and fibers
CCK-like immunoreactive cell bodies have been identified in the
DRNR that are situated both rostral and caudal to the 5-HT-
immunoreactive perikarya.
78
In addition, CCK receptors with
a profile similar to the CCK
A
subtype have been characterized on
DRN 5-HT-containing cells of the rat.
79
CCK has been found also to
be colocalized with 5-HT in the medulla of the rat.
80
NT-containing neurons and fibers
Neuronal cell bodies containing NT along with NT-containing
nerve terminals have been visualized in the DRNR of the rat.
81,82
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Moreover, the distribution of NT receptors in the DRN agrees with
the immunohistochemical localization of NT-immunoreactive
nerve terminals.
83
SOM-containing neurons and fibers
Double SOM-immunoreactive and [
3
H]-5-HT radiolabeled
neurons have been characterized in the DRNR and DRND of the
rat.
84
SOM mRNA in the same subregions has been found to be
expressed also by a separate non-serotonergic cell population.
85
A
moderate density of SOM receptors and SOM-immunoreactive
fibers has been found, in addition, in the DRN of the sheep and the
cat, respectively.
86,87
GH and GHRH containing neurons and fibers
Data on the distribution of growth hormone (GH) and growth
hormone-releasing hormone (GHRH) immunoreactive perikarya,
fibers and receptors in the DRN are not available to date.
Enkephalin-containing neurons and fibers
Uhl et al.
81
initially visualized enkephalin immunoreactive
perikarya, fibers and nerve terminals in the raphe nuclei of the rat.
Soon after, Moss et al.
88
determined the distribution of leu-
enkephalin immunoreactive cells in the DRN of the cat. Enkephalin
perikarya were found along the midline of the DRN but extended
also laterally. Some DRN neurons predominantly located on the
midline of the nucleus contain both 5-HT and leu-enkephalin.
89,90
More recently, immunoreactive cell bodies have been observed in
the DRN of the cat that contain met-enkephalin and beta endorphin
in addition to leu-enkephalin.
91
Interestingly, leu-enkephalin neurons have been characterized
in the DRN of the rat that send projection fibers to the central
amygdaloid nucleus bilaterally with an ipsilateral dominance.
91
NPY-containing neurons and fibers
NPY is a widely distributed neuropeptide in the central nervous
system of the rat. The main source of NPY in the brain is the
hypothalamus, particularly the suprachiasmatic nucleus, the
arcuate nucleus, the paraventricular nucleus and the dorsomedial
nucleus.
92
In addition, NPY-immunoreactive neurons have been
characterized within the rostral part (DRNR) of the DRN where they
have been proposed to innervate CRF-expressing cells.
22
Firing pattern of 5-HT and non-5-HT neurons of the DRN
during the sleep-wake cycle
The functional activity of DRN 5-HT neurons has been studied in
in vitro preparations, urethane-anesthetized rats, and freely-
moving cats. Spontaneously active immunohistochemically iden-
tified 5-HT cells recorded in a slice preparation of the rat DRN are
characterized by slow, rhythmic activity, a broad action potential,
a large afterhyperpolarization potential, and a decrease of their
firing rate in response to the stimulation of the 5-HT
1A
somato-
dendritic receptor.
93,94
Using a similar experimental approach, Kirby et al.
95
charac-
terized the electrophysiological properties of 5-HT and non-5-HT
cells located predominantly in the DRNV and the DRNI clusters of
the rat. 5-HT neurons had a significantly lower firing rate, a shorter
time constant (tau, an estimate of membrane capacitance), and
a slower onset of the afterhyperpolarization potential than non-5-
HT cells.
Allers and Sharp
96
successfuly identified the neurochemical
and morphological properties of 5-HT-containing cells and GABA-
containing cells of the DRN in urethane-anesthetized rats. The
study authors characterized a group of neurons immunoreactive
for 5-HT or both 5-HT and tryptophan hydroxylase that fired broad
spikes with low frequency and a highly regular pattern. Systemic
administration of 8-OHD-PAT markedly inhibited the firing rate of
these neurons. A fast firing population of cells was described, in
addition, that was immunoreactive for GAD and immunonegative
for tryptophan hydroxylase. All these neurons had significantly
faster firing rates and narrower spikes widths than the 5-HT-
containing cells.
Electrophysiological recordings in unanesthetized animals
allowed McGinty and Harper
97
and Trulson and Jacobs
2
to charac-
terize the activity of DRN 5-HT neurons across the sleep-wake
cycle. During quiet waking 5-HT neurons fire in a slow and regular
fashion. During active waking the neuronal activity shows a 30–50%
increase. As the animal enters SWS the firing rate decreases to 50%
of the quiet waking state and is no longer regular. Finally, during
REMS there is a further decrease or even a cessation of neuronal
activity.
Notwithstanding the above, numerous presumed DRN 5-HT
neurons do not adhere to this pattern of discharge. In this respect,
Sakai et al.
98,99
have described two types of presumed DRN 5-HT
cells according to their regular (type I) or irregular (type II) firing
pattern. Type I neurons are evenly distributed within the DRN and
their activity is correlated to the behavioral state. However, some of
these cells show a complete suppression of discharge prior to the
appearance of ponto-geniculo-occipital (PGO) waves, whereas
other neurons cease firing only during REMS. Furthermore, a small
percentage of type I cells that show a reduction of their firing rate
during SWS and REMS, maintain a relatively high level of tonic
activity.
A subgroup of type II cells located in the middle portion of the
DRN discharge at a high rate during active waking; their activity
decreases during quiet waking and is suppressed during SWS and
REMS. Moreover, type II cells have been described in the most
rostral and dorsal portion of the DRN that discharge at their highest
rate during SWS and become silent during both W and REMS.
Thus, in addition to marked differences in cellular morphology,
expression of other neurotransmitters, and afferent and efferent
connections presumed DRN 5-HT neurons show differences in their
firing pattern during the sleep-wake cycle.
Operational characteristics of the 5-HT receptors
The 5-HT receptors can be classified into seven classes, desig-
nated 5-HT
1–7
. The classification of receptors is based upon
sequence similarity and second messenger pathways coupled to
receptor activation. The 5-HT
1
, 5-HT
2
, 5-HT
3
and 5-HT
5
classes
consist of five (5-HT
1A-B-D-E-F
), three (5-HT
2A-B-C
), and two (5-HT
3A-
B
, 5-HT
5A-B
) subtypes, respectively, whereas the 5-HT
4
, 5-HT
6
, and
5-HT
7
classes have at present one subtype each.
100–102
Except for
the 5-HT
3
receptor, all other 5-HT receptors are structurally related
to the superfamily of G-protein-coupled receptors.
The 5-HT
1A
receptor
Early studies proposed that the 5-HT
1A
receptor is exclusively
located on the soma and the dendrites (somatodendritic autor-
eceptor) of 5-HT neurons and at postsynaptic sites (outside the
DRN). More recently, the 5-HT
1A
receptor has been found to be
expressed also by non-5-HT cells of the DRN.
36,95
According to
Kirby et al.,
95
40–50% of immunohistochemically identified non-5-
HT neurons express the 5-HT
1A
receptor. On the other hand, Day
J.M. Monti / Sleep Medicine Reviews 14 (2010) 307–317 313
Author's personal copy
et al.
36
have proposed, on the basis of an in situ hibridization study,
that only 10–15% of the non-5-HT cells express the 5-HT
1A
receptor,
and that most of them are GABA-containing neurons.
Four classes of G-protein are known: Gs (activates adenylyl
cyclase), Gi (inhibits adenylyl cyclase), Gq (activates phospholipase-
C), and Go (inhibits voltage-dependent Ca
2þ
and K
þ
channels). The
5-HT
1A
receptor couples to Gi/o proteins, with Gi protein activation
leading to an inhibition of adenylyl cyclase. On the other hand,
activation of the 5-HT
1A
receptor leads to closing of Ca
2þ
channels
via Go protein and opening of K
þ
channels. Stimulation of the
somatodendritic 5-HT
1A
receptor inhibits the firing rate of seroto-
nergic neurons, whereas activation of the postsynaptic receptor
induces inhibitory responses on target structures. Activation of
postsynaptic 5-HT
1A
receptors expressed by GABAergic cells located
in the DRN would be expected to indirectly facilitate the activity of
5-HT neurons. In this respect, Koyama et al.
103
have shown that 5-HT
reduced the frequency of miniature inhibitory postsynaptic
currents arising from attached GABAergic presynaptic terminals
recorded in dissociated rat basolateral amygdala nuclei. The sero-
tonergic effect was reproduced by the 5-HT
1A
receptor agonist 8-
OH-DPAT and prevented by the 5-HT
1A
antagonist spiperone.
Brain areas rich in 5-HT
1A
receptors include the telencephalon
[cerebral cortex, limbic system (medial and lateral septal nuclei,
hippocampal formation, amygdala)]; the diencephalon [thalamus,
hypothalamus (ventromedial and supraoptic nuclei)]; the mesen-
cephalon (VTA/SNc, LDT/PPT); and the rhombencephalon (DRN,
MRN, raphe pallidus, raphe obscurus, LC).
104–108
The 5-HT
1B
receptor
The 5-HT
1B
receptor is linked to the inhibition of adenylate
cyclase and was initially proposed to be located at presynaptic
(5-HT axon terminals) and postsynaptic (outside the DRN) sites.
The 5-HT
1B
receptor has been characterized also in the ventro-
medial DRN where it is expressed by non-5-HT cells.
109
The
5-HT
1B
receptor is involved in the regulation of the synaptic
release of 5-HT and of other neurotransmitters, including ACh, NE,
GABA and GLU, which is indicative of its role as auto- and heter-
oreceptor, respectively.
Mapping of the 5-HT
1B
receptor mRNA and its visualization by
autoradiography tends to indicate that its distribution is widespread
in the central nervous system of several species, including man.
Accordingly, the 5-HT
1B
receptor has been found in the telencephalon
[cerebral cortex, limbic system (amygdala, hippocampus, lateral
septal nucleus), basal ganglia]; the diencephalon [thalamus, hypo-
thalamus (lateral preoptic area, anterior, lateral and dorsal hypotha-
lamic areas, suprachiasmatic nucleus)]; the mesencephalon (central
gray, interpeduncular nucleus, VTA/SNc); and the rhombencephalon
(DRN/MRN, LC, raphe magnus nucleus, cerebellum).
110116
The 5-HT
2A
and the 5-HT
2C
receptors
The 5-HT
2A
and the 5-HT
2C
receptors have striking amino acid
homology. They are primarily coupled to Gq protein and their
actions are mediated by the activation of phospholipase C, with
a resulting depolarization of the host cell.
101,117
Receptors of the 5-
HT
2
subfamily are located within postsynaptic structures,
predominantly on proximal and distal dendritic shafts.
The 5-HT
2A
and the 5-HT
2C
receptors have been characterized in
the telencephalon [cerebral cortex, olfactory system, limbic system
(septal nuclei, hippocampal formation, amygdala), basal ganglia];
the diencephalon [thalamus, hypothalamus (medial and lateral
preoptic areas, ventromedial and dorsomedial nuclei, mammillary
nucleus)]; the mesencephalon (central gray, VTA/SNc); and the
rhombencephalon (LDT/PPT, medial pontine reticular formation
(mPRF), DRN/MRN, LC).
118–124
It should be mentioned that 5-HT
neurons of the DRN do not express 5-HT
2A
or 5-HT
2C
receptors. The
serotonin 5-HT
2A
and 5-HT
2C
receptor-containing neurons are
predominantly GABAergic interneurons and projection
neurons.
123,125–127
Thus, activation of 5-HT
2A
and 5-HT
2C
receptors
expressed by GABAergic cells located in the DRN would result in the
decrease 5-HT neuronal firing rate.
The 5-HT
3
receptor
The 5-HT
3
receptor is not coupled to G proteins. It directly
activates a 5-HT-gated cation channel, which leads to the depo-
larization of a variety of cells. As a result, there is an increase in the
release of DA, NE, GABA, GLU, ACh and 5-HT at central sites.
101
The 5-HT
3
receptor is present in cortical and subcortical struc-
tures including the cerebral cortex, olfactory system, limbic system
(hippocampus, amygdala), basal ganglia, central gray, mesence-
phalic reticular formation, and DRN.
128–133
At the DRN level
the 5-HT
3
receptor is expressed, among others, by glutamatergic
interneurons.
132
Thus, activation of the 5-HT
3
receptor expressed
by glutamatergic interneurons located in the DRN would be
expected to induce the activation of 5-HT neurons.
The 5-HT
7
receptor
The 5-HT
7
receptor has been cloned from a variety of species,
including the rat, and has been shown to be positively coupled to
adenylate cyclase via G
s
-proteins. The 5-HT
7
receptor has been
discretely localized in both terminal regions and cell body regions
involved in the regulation of SWS, REMS, and W in the rat, including
the pyramidal neurons of the cerebral cortex, the anterior thal-
amus, the hippocampus (fields CA1 and CA3 of Ammon’s horns),
the suprachiasmatic nucleus, the pontine nuclei, the brain stem
reticular formation, the DRN and the MRN.
134–138
5-HT
7
receptors
in the DRN are not localized to serotonergic neurons and conse-
quently do not subserve an autoreceptor function.
139
In this respect,
Roberts et al.
140,141
and Glass et al.
142
have proposed, on the basis of
a series of functional studies, that 5-HT
7
receptors in the DRN are
localized, at least in part, to GABAergic cells and terminals. Acti-
vation of 5-HT
7
receptors expressed by GABAergic interneurons
would result in the inhibition of 5-HT-containing cells.
Conclusions
It has been established that a number of neuroanatomical
structures corresponding to the arousal system are located in the
brain stem. They include, among others, the DRN which provides the
principal source of serotonergic innervation of the cerebral cortex,
amygdala, BFB, thalamus, preoptic and hypothalamic areas, raphe
nuclei, LC and pontine reticular formation. It is currently accepted
that inhibitory and facilitatory neurotransmitter systems project to
the DRN and regulate the activity of 5-HT neurons during the sleep-
wake cycle. Among the former are the GABAergic and the MCH-ergic
systems that can promote REMS by inhibiting 5-HT neurons. On the
other hand, the noradrenergic, dopaminergic, histaminergic,
cholinergic and orexinergic systems provide excitatory afferents to
DRN 5-HT cells that would induce the opposite effect.
The presence of 5-HT
1B
, 5-HT
2A/2C
, 5-HT
3
and 5-HT
7
receptors
within the boundaries of the DRN, expressed by GABAergic and
glutamatergic interneurons, tends to indicate the existence of
a supplemental mechanism in the control of 5-HT neurons func-
tional activity. Contributing to the modulation of 5-HT neurons are
also several different types of neuropeptides. The unique properties
and function of 5-HT cells would depend in great measure on the
influence of afferent inputs, 5-HT receptor subtypes expressed by
J.M. Monti / Sleep Medicine Reviews 14 (2010) 307–317314
Author's personal copy
DRN interneurons and neuropeptides that are either synthesized by
distinct cells in the DRN or coexpressed with other neurochemicals.
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Practice points
1. Serotonergic neurons of the DRN appear in topograph-
ically organized groups that differ in their morphological
characteristics, cellular properties, and afferent and
efferent connections.
2. Inhibitory and facilitatory systems project to the DRN
that contribute to the regulation of 5-HT neuron’s func-
tional activity.
3. 5-HT receptor subtypes expressed by DRN interneurons
have been shown to play a role in the regulation of the
behavioral state.
Research agenda
1. Studies aimed at determining the presence of 5-HT
4
,5-
HT
5
and 5-HT
6
receptors within the boundaries of the
DRN.
2. Evaluation of the influence of neuropeptides located in
the DRN on 5-HT cells’ functional activity during the
sleep-wake cycle.
3. Research is needed to better understand the influence of
5-HT receptor subtypes on the structures involved in the
induction and maintenance of REMS.
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